NRIF3, a novel co-activator for nuclear hormone receptors

ABSTRACT

Nucleic acids encoding NRIF3 are described. Polypeptides having amino acid sequences of NRIF3 proteins are also provided. A method is also provided for isolating and cloning NRIF3 cDNA. NRIF3 is useful in development/implementation of high throughput screens to identify novel thyroid hormone receptor (TR) and retinoid X receptor (RXR) agonists and antagonists. Methods are also provided for identifying compounds that directly interfere with the interaction of NRIF3 and TR or RXR. Finally, therapies based on modulation of NRIF3 activity are disclosed.

PRIORITY

This application is a continuation of U.S. patent application Ser. No.09/662,052, filed Sep. 15, 2000, now U.S. Pat. No. 6,639,064, whichclaims priority under 35 U.S.C. §119 from provisional patent applicationSer. No. 60/154,347, filed Sep. 17, 1999, both of which are herebyincorporated by reference in their entireties.

The research leading to the present invention was supported, in part, byNational Institutes of Health Grants No. DK09581 and No. DK16636-27.Accordingly, the United States Government has certain rights in theinvention.

FIELD OF THE INVENTION

This invention relates to a novel nuclear hormone receptor co-activator.The invention further relates to high throughput screening assays withthese receptors, as well as utilization of the co-activator fordeveloping therapeutic measures for human diseases.

BACKGROUND OF THE INVENTION

Nuclear hormone receptors are ligand-regulated transcription factorsthat play diverse roles in cell growth, differentiation, development,and homeostasis. The nuclear receptor superfamily has been divided intotwo sub-families: the steroid receptor family and the thyroidhormone/retinoid (non-steroid) receptor family (51). The steroidreceptor family includes receptors for glucocorticoids (GR),mineralcorticoids (MR), progestins (PR), androgens (AR) and estrogens(ERs) (51). The non-steroid receptor family includes receptors forthyroid hormones (TRs), retinoids (RARs and RXRs), 1,25-(OH)₂ vitamin D(VDR), prostanoids (PPARs) as well as many orphan receptors whoseligands (if any) remain to be defined (49, 51). Members of the nuclearreceptor superfamily share common structural and functional motifs.Nevertheless, an important difference exists between the twosub-families. Steroid receptors primarily act as homodimers by bindingto their cognate palindromic hormone response elements (HREs) (77, 78).In contrast, members of the non-steroid receptor family can bind to DNAas monomers, homodimers, and heterodimers (25, 78). Their correspondingHREs are also complex, and can be organized as direct repeats, invertedrepeats, and everted repeats (49). Therefore, the combination ofheterodimerization and HRE complexity provides the potential to generateenormous diversity in receptor-mediated regulation of target geneexpression.

Structural and functional studies indicate that the ligand bindingdomain (LBD) of many members of the thyroid hormone/retinoid receptorfamily harbors diverse functions. In addition to ligand binding, the LBDalso plays roles in mediating receptor dimerization, hormone-dependenttransactivation, and in the case of TR and RAR, ligand-relieved genesilencing (54, 61). The carboxyl-terminal helix of the LBD has beenimplicated in playing an important role in ligand-dependentconformational changes and transactivation (6, 9, 21, 43). Although ithas been suggested that an activation function (AF-2) resides in thisC-terminal helix, recent studies indicate that AF-2 results from aligand-induced conformational change involving diverse areas of the LBD(23, 66). Thus, ligand binding serves to switch the receptor from onefunctional state (e.g. inactive or silencing) to another (e.g.transactivation).

Although much has been learned from studying the structure and functionof these receptors, the detailed molecular mechanism(s) oftranscriptional regulation by these receptors is not well understood.Efforts to understand the molecular mechanism of transcriptionalrepression by unliganded TRs and RARs have led to the description (12)and isolation of putative co-repressor proteins SMRT and N-CoR, whichinteract with the LBD of these receptors in the absence of their ligands(15, 36). The recent discovery that both SMRT and N-CoR form complexeswith Sin3 and a histone deacetylase suggests that chromatin remodelingby histone deacetylation may play a role in receptor-mediatedtranscriptional repression (33, 55).

In a somewhat parallel approach, the identification of co-activators hasrecently received extensive experimental attention in order to elucidatethe molecular mechanism(s) of transcriptional activation by nuclearreceptors (27). Identified co-activator proteins primarily belong to twogroups: the SRC-1 family and the CBP/p300 family. The SRC-1 familyincludes SRC-1/NCoA-1 (37, 58, 74), and the related proteinsGRIP1/TIF2/NCoA-2 (34, 35, 74, 79), and AMB1/p/CIP/ACTR/RAC3/TRAM-1 (2,14, 44, 73, 74). The second group of co-activators includes CBP and itshomolog p300, which not only influence the activity of nuclear receptors(13, 31, 37), but also functionally interact with many transcriptionfactors such as CREB (3, 16, 40, 46), the Stats (10, 87), AP1 (4, 7),and p53 (28, 45). There are also co-activator proteins that do notbelong to these two groups, such as ARA70 (85), PGC-1 (60), and therecently-reported RNA co-activator SRA (41). Members of both the SRC-1family and CBP/p300 family have been shown to possess histoneacetyltransferase (HAT) activities (8, 14, 57, 69), suggesting thatchromatin remodeling by histone acetylation is an important mechanisminvolved in transcriptional activation by ligand-bound nuclearreceptors.

Interaction of members of the SRC-1 and CBP/p300 families with nuclearreceptors occurs through conserved LxxLL (SEQ ID NO:1) motifs (32),which interact with a hydrophobic cleft in the receptor LBD formed as aresult of conformational changes mediated by ligand binding (19, 23,56). In the sequence, x refers to any amino acid. SRC-1/NCoA-1 andGRIP1/TIF2 contain three LxxLL regions or boxes (referred to as LXDs orNR boxes) that differentially interact with nuclear receptors so thatdifferent nuclear receptors functionally utilize different LxxLL boxes(19, 52). Thus, ER utilizes the second LxxLL box of SRC-1/NCoA-1 whilePR utilizes both the first and second LxxLL boxes for optimalinteraction. In contrast, TR and RAR require both the second and thirdLxxLL boxes for optimal interaction (52). The specificity of receptorrecognition by the different LxxLL boxes of SRC-1/NCoA-1 is primarilymediated by eight amino acid residues C-terminal to the LxxLL motifrather than by the two amino acids (xx) within the motif itself. Thus,while members of the SRC-1 family are capable of interacting with manynuclear receptors, the molecular detail of such interactions differs foreach receptor in the number or combination of LxxLL boxes utilized aswell as in the critical amino acid residues surrounding the LxxLLmotifs.

While much has been learned from the study of known co-activators, anumber of key mechanistic questions remain to be answered. For example,many nuclear receptors can recognize common DNA elements, (25, 49, 51),while not all are capable of regulating genes containing those elements(20, 47, 65). Thus, how native target genes containing such elements areselectively regulated by specific receptors is a very important butpoorly-understood problem. Although the various LxxLL boxes of SRC-1 andGRIP1 show differential receptor preference (19, 52), theseco-activators are unlikely to play a primary role in mediating effectsthat are receptor specific since they appear to interact with allligand-bound nuclear hormone receptors. Thus, the detailed molecularmechanism(s) underlying receptor-specific regulation of gene expressionremains to be elucidated. Whether co-activator(s) might contribute tothis specificity is currently unknown.

SUMMARY OF THE INVENTION

The present invention discloses an isolated nucleic acid moleculecomprising a sequence that encodes a functional NRIF3 nuclear hormonereceptor co-activator, where the NRIF3 binds in a ligand dependentmanner to thyroid hormone receptor (TR) and retinoid X receptor (RXR),but does not interact with retinoic acid receptor (RAR), vitamin Dreceptor (VDR), progesterone receptor (PR), glucocorticoid receptor(GR), and estrogen receptor (ER) in a yeast two hybrid assay system orin vitro, or both, and where the nucleic acid encodes a polypeptide thatcontains an LxxIL (SEQ ID NO:2) module in its C-terminal domain.

A hybridizable nucleic acid, of at least twenty bases, which has asequence as depicted in SEQ ID NO:3 (FIG. 2) also is contemplated by thepresent invention.

The present invention also contemplates an isolated functional NRIF3nuclear hormone receptor co-activator, where the NRIF3 binds in a liganddependent manner to thyroid hormone receptor (TR) and retinoid Xreceptor (RXR), but does not interact with retinoic acid receptor (RAR),vitamin D receptor (VDR), progesterone receptor (PR), glucocorticoidreceptor (GR), and estrogen receptor (ER) in a yeast two hybrid assaysystem or in vitro, or both, where the polypeptide contains an LxxIL(SEQ ID NO:2) module in its C-terminal domain.

A method for identifying a compound that modulates NFIR3 interactionwith a nuclear hormone receptor is also contemplated by the presentinvention. The method comprises detecting modulation of the interactionof NFIR3 and TR or RXR in the presence of the compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Hormone-dependent interaction of NRIF3 with the ligand bindingdomain (LBD) of TR. Induction of β-galactosidase activity by thyroidhormone (T3) was measured in the yeast strain EGY48 transformed with abait vector expressing LexA-cTRα LBD and the prey plasmid expressingNRIF3 fused to the B42 activation domain (29). The bait LexA alone wasused as the negative control. The prey B42-Trip1 was used as thepositive control.

FIG. 2. Nucleotide (SEQ ID NO:3) and deduced amino acid (SEQ ID NO:4)sequences of NRIF3. Only part of the cDNA sequence is shown. A putativenuclear localization signal (KRKK; SEQ ID NO:5) is underlined. Theputative LxxLL (SEQ ID NO:1) motif is shown with a double underline.NRIF3 and the β3-endonexin long form (EnL) share 95% identity. Theydiffer only in the C-terminus where the last 16 amino acids (dotunderlined) in NRIF3 is replaced with 9 different amino acids(GQPQMSQPL; SEQ ID NO:6) in the β3-endonexin long form. The short formof β3-endonexin consists of 111 amino acids and is 100% identical to thefirst 111 amino acids of NRIF3 or the β3-endonexin long form.

FIG. 3. NRIF3 is a nuclear protein. HeLa cells were transfected with aexpression vector for GFP (left panel) or GFP-NRIF3 (right panel). Thecellular location of the expressed proteins was visualized byfluorescence microscopy.

FIG. 4. Characterization of the NRIF3 interaction with nuclear receptorsin vitro. ³⁵S-labeled full length receptor (cTRα, hRARα, hRXRα, hVDR,hPR, hGR, or hER) was incubated with affinity purified GST control orGST-NRIF3 linked to glutathione-agarose beads. The binding was performedin the absence (−) or presence (+) of cognate ligands as described inMaterials and Methods. After incubation and washing, the bound receptorswere analyzed in 10% SDS-PAGE and detected by autoradiography. The inputlane in each binding assay represents 5% of the total ³⁵S-labeledreceptor used in each incubation. GST-RXR was used as a positive controlfor RAR binding.

FIGS. 5A and 5B. NRIF3 enhances TR-mediated transactivation in vivo.HeLa cells were transfected with a vector expressing cTRα, and theIR-ΔMTV-CAT reporter (A) or the GH-TRE-tk-CAT reporter (B) in thepresence (filled columns) or the absence (hatched columns) of 1 μM T3.The vector expressing NRIF3 or the empty control vector wereco-transfected to examine the effect of NRIF3 on TR-mediated activation.In (A), the effect of CBP was compared to that of NRIF3.

FIGS. 6A, 6B, and 6C. NRIF3 functions as a co-activator for RXR but notRAR. (A) NRIF3 potentiates the activity of endogenous RXR(s) but notRAR(s). HeLa cells were transfected with the IR-ΔMTV-CAT reporter(without any receptor expression vector) to examine the activation byendogenous retinoid receptors. The NRIF3 expression vector or the emptycontrol vector were co-transfected to examine the effect of NRIF3 on theactivity of endogenous RXR(s) or RAR(s). Relative CAT activity wasdetermined in the presence (filled columns) or absence (hatched columns)of indicated ligands (1 μM). (B), (C) NRIF3 potentiates the activity ofexogenously expressed RXR. A vector expressing hRXRα was co-transfectedinto HeLa cells with the IR-ΔMTV-CAT reporter (B) or the DR1-ΔMTV-CATreporter (C), in the presence (filled columns) or absence (hatchedcolumns) of indicated ligands (1 μM). The effect of NRIF3 onRXR-mediated transactivation was similarly examined as in (A).

FIGS. 7A, 7B, and 7C. NRIF3 does not potentiate the activity of GR, PR,ER, or VDR. HeLa cells were transfected with the following CAT reportersand appropriate receptor expression vectors: GRE/PRE-tk-CAT and rGR orhPR (A), ERE-ΔMTV-CAT and hER (B), VDRE-ΔMTV-CAT and hVDR (C). Cellswere incubated in the presence (filled columns) or absence (hatchedcolumns) of 100 mM dexamethathone for GR, progesterone for PR, estradiolfor ER, and 1,25-(OH)₂-VitD3 for VDR. Co-transfection of NRIF3 was foundto have little effect on the activity of these receptors.

FIGS. 8A and 8B. The C-terminal domain of NRIF3 is essential for theinteraction with liganded TR or RXR. (A) schematic comparison of NRIF3with EnS and EnL. EnS is 100% identical to the first 111 amino acids ofNRIF3 or EnL (open box). The region from amino acid 112 to 161 is 100%identical between NRIF3 and EnL (dotted box). NRIF3 and EnL differ intheir C-terminus (16 amino acids in NRIF3, hatched box; and 9 aminoacids in EnL, filled box). The positions of the LxxLL (SEQ ID NO:1)motif and a putative nuclear localization signal (KRKK; SEQ ID NO:5) arealso indicated. (B) NRIF3 (N), EnS (S), or EnL (L) was examined forinteraction with LexA-TR or LexA-RXR in a yeast two-hybrid assay asdescribed in Materials and Methods. The assays were performed in theabsence (hatched columns) or the presence (filled columns) of 1 μM T3(for TR) or 9-cis RA (for RXR).

FIG. 9. The LxxLL (SEQ ID NO:1) motif of NRIF3 is required for optimuminteraction with TR and RXR. Wild type NRIF3 (WT) or the L9A NRIF3mutant (L9A) was examined for interaction with LexA-TR or LexA-RXR in ayeast two-hybrid assay as described in Materials and Methods.β-galactosidase activities were determined in the absence (filledcolumns) or presence (dotted columns) of cognate ligands (1 μM T3 forTR, 1 μM 9-cis RA for RXR).

FIG. 10. A hypothetical model of interaction of the NRIF3 C-terminaldomain (NCD) and the liganded LBD. The docking of the C-terminal helixof NRIF3 which contains an LxxIL (SEQ ID NO:2) module to theligand-bound LBDs was carried out as described in Materials and Methods.The NCD/TR LBD model is shown here as an example. The side chains of thetwo leucines (green) and one isoleucine (cyan) of the LxxIL core fitwithin a hydrophobic groove (salmon) on the surface of the liganded LBD.A similar modeling procedure was carried out using an LxxLL (SEQ IDNO:1) box of SRC-1 (result not shown). Putative binding energies (−21kcal/mol for the NCD, and −18 kcal/mol for the LxxLL box of SRC-1) werecalculated as described in Materials and Methods. See text for details.

FIG. 11. Interaction of the NCD with the receptor LBDs and the role ofthe LxxIL motif The wild type NCD (WT) or the NCD mutant form (Mut) inwhich the three core hydrophobic residues of the LxxIL (SEQ ID NO:2)motif (two leucines and one isoleucine) are changed into alanines, wasexamined for interaction the LBDs of TR, RXR, and RAR in a yeasttwo-hybrid assay as described in Materials and Methods. β-galactosidaseactivities were determined in the absence (empty columns) or presence(dotted columns) of cognate ligands. The prey expressing B42 alone wasused as a negative control.

DETAILED DESCRIPTION OF THE INVENTION

Many nuclear receptors are capable of recognizing similar DNA elements.The molecular event(s) underlying the functional specificity of thesereceptors (in regulating the expression of their native target genes) isa very important question that remains poorly understood. The presentinvention is based, in part, on the cloning and analysis of a novelnuclear receptor co-activator (designated as NRIF3) that exhibits adistinct receptor specificity. Fluorescence microscopy shows that NRIF3localizes to the cell nucleus. Yeast two-hybrid and/or in vitro bindingassays indicate that NRIF3 specifically interacts with TR (thyroidhormone receptor) and RXR (retinoid X receptor) in a ligand-dependentfashion, but does not bind to RAR (retinoic acid receptor), VDR (vitaminD receptor), PR (progesterone receptor), GR (glucocorticoid receptor),or ER (estrogen receptor). Functional studies show that NRIF3significantly potentiates TR- and RXR-mediated transactivation in vivowhile little effect is observed for other examined nuclear receptors.Domain and mutagenesis analyses indicate that a novel C-terminal domainin NRIF3 plays an essential role in its specific interaction withliganded TR and RXR, while the N-terminal LxxLL (SEQ ID NO:1) motifplays a minor role in allowing optimum interaction. Computer modelingand subsequent experimental analysis suggest that the C-terminal domainof NRIF3 directly mediates interaction with liganded receptors throughan LxxIL (SEQ ID NO:2)(a variant of the canonical LxxLL; SEQ ID NO:1)module, while other part of the NRIF3 protein may still play a role inconferring its receptor specificity. In the sequence, x refers to anyamino acid. Identification of a co-activator with such a unique receptorspecificity may provide new insight into the molecular mechanism(s) ofreceptor-mediated transcriptional activation as well as the functionalspecificity of nuclear receptors.

The use of italics indicates a nucleic acid molecule (e.g., NRIF3, cDNA,gene, etc.); normal text indicates the polypeptide or protein.

Genes Encoding NRIF3 Proteins

The present invention provides a gene encoding a NRIF3 of the invention,including a full length, or naturally occurring form of NRIF3, genomicNRIF3, splice variants of NRIF3 and any antigenic fragments thereof fromany human source.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

A “vector” is a recombinant nucleic acid construct, such as plasmid,phage genome, virus genome, cosmid, or artificial chromosome, to whichanother DNA segment may be attached. In a specific embodiment, thevector may bring about the replication of the attached segment, e.g., inthe case of a cloning vector. A “replicon” is any genetic element (e.g.,plasmid, chromosome, virus) that functions as an autonomous unit of DNAreplication in vivo, i.e., it is capable of replication under its owncontrol.

A “cassette” refers to a segment of DNA that can be inserted into avector at specific restriction sites. The segment of DNA encodes apolypeptide of interest, and the cassette and restriction sites aredesigned to ensure insertion of the cassette in the proper reading framefor transcription and translation.

A cell has been “transfected” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. A cell has been “transformed”by exogenous or heterologous DNA when the transfected DNA is expressedand effects a function or phenotype on the cell in which it isexpressed.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear (e.g., restrictionfragments) or circular DNA molecules, plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

A “polynucleotide” or “nucleotide sequence” is a series of nucleotidebases (also called “nucleotides”) in DNA and RNA, and means any chain oftwo or more nucleotides. A nucleotide sequence typically carries geneticinformation, including the information used by cellular machinery tomake proteins and enzymes. These terms include double or single strandedgenomic and cDNA, RNA, any synthetic and genetically manipulatedpolynucleotide, and both sense and anti-sense polynucleotide (althoughonly sense stands are being represented herein). This includes single-and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNAhybrids, as well as “protein nucleic acids” (PNA) formed by conjugatingbases to an amino acid backbone. This also includes nucleic acidscontaining modified bases, for example thio-uracil, thio-guanine andfluoro-uracil.

The polynucleotides herein may be flanked by natural regulatory(expression control) sequences, or may be associated with heterologoussequences, including promoters, internal ribosome entry sites (IRES) andother ribosome binding site sequences, enhancers, response elements,suppressors, signal sequences, polyadenylation sequences, introns, 5′-and 3′-non-coding regions, and the like. The nucleic acids may also bemodified by many means known in the art. Non-limiting examples of suchmodifications include methylation, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, andinternucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters,phosphoroamidates, carbamates, etc.) and with charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.). Polynucleotides maycontain one or more additional covalently linked moieties, such as, forexample, proteins (e.g., nucleases, toxins, antibodies, signal peptides,poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.),chelators (e.g., metals, radioactive metals, iron, oxidative metals,etc.), and alkylators. The polynucleotides may be derivatized byformation of a methyl or ethyl phosphotriester or an alkylphosphoramidate linkage. Furthermore, the polynucleotides herein mayalso be modified with a label capable of providing a detectable signal,either directly or indirectly. Exemplary labels include radioisotopes,fluorescent molecules, biotin, and the like.

The term “host cell” means any cell of any organism that is selected,modified, transformed, grown, or used or manipulated in any way, for theproduction of a substance by the cell, for example the expression by thecell of a gene, a DNA or RNA sequence, a protein or an enzyme. Hostcells can further be used for screening or other assays, as describedinfra.

Proteins and enzymes are made in the host cell using instructions in DNAand RNA, according to the genetic code. Generally, a DNA sequence havinginstructions for a particular protein or enzyme is “transcribed” into acorresponding sequence of RNA. The RNA sequence in turn is “translated”into the sequence of amino acids which form the protein or enzyme. An“amino acid sequence” is any chain of two or more amino acids. Eachamino acid is represented in DNA or RNA by one or more triplets ofnucleotides. Each triplet forms a codon, corresponding to an amino acid.For example, the amino acid lysine (Lys) can be coded by the nucleotidetriplet or codon AAA or by the codon AAG. (The genetic code has someredundancy, also called degeneracy, meaning that most amino acids havemore than one corresponding codon.) Because the nucleotides in DNA andRNA sequences are read in groups of three for protein production, it isimportant to begin reading the sequence at the correct amino acid, sothat the correct triplets are read. The way that a nucleotide sequenceis grouped into codons is called the “reading frame.”

A “coding sequence” or a sequence “encoding” an expression product, suchas a RNA, polypeptide, protein, or enzyme, is a nucleotide sequencethat, when expressed, results in the production of that RNA,polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodesan amino acid sequence for that polypeptide, protein or enzyme. A codingsequence for a protein may include a start codon (usually ATG) and astop codon.

The term “gene”, also called a “structural gene” means a DNA sequencethat codes for or corresponds to a particular sequence of amino acidswhich comprise all or part of one or more proteins or enzymes, and mayor may not include regulatory DNA sequences, such as promoter sequences,which determine for example the conditions under which the gene isexpressed. Some genes, which are not structural genes, may betranscribed from DNA to RNA, but are not translated into an amino acidsequence. Other genes may function as regulators of structural genes oras regulators of DNA transcription.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as regions(consensus sequences) responsible for the binding of RNA polymerasemachinery.

A coding sequence is “under the control of” or “operatively associatedwith” transcriptional and translational control sequences in a cell whenRNA polymerase transcribes the coding sequence into mRNA, which is thentrans-RNA spliced (if it contains introns) and translated into theprotein encoded by the coding sequence.

The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing a protein by activating the cellular functions involved intranscription and translation of a corresponding gene or DNA sequence. ADNA sequence is expressed in or by a cell to form an “expressionproduct” such as a protein. The expression product itself, e.g. theresulting protein, may also be said to be “expressed” by the cell. Anexpression product can be characterized as intracellular, extracellularor secreted. The term “intracellular” means something that is inside acell. The term “extracellular” means something that is outside a cell. Asubstance is “secreted” by a cell if it appears in significant measureoutside the cell, from somewhere on or inside the cell.

The term “transfection” means the introduction of a foreign nucleic acidinto a cell. The term “transformation” means the introduction of a“foreign” (i.e. extrinsic or extracellular) gene, DNA or RNA sequence toa host cell, so that the host cell will express the introduced gene orsequence to produce a desired substance, typically a protein or enzymecoded by the introduced gene or sequence. The introduced gene orsequence may also be called a “cloned” or “foreign” gene or sequence,may include regulatory or control sequences, such as start, stop,promoter, signal, secretion, or other sequences used by a cell's geneticmachinery. The gene or sequence may include nonfunctional sequences orsequences with no known function. A host cell that receives andexpresses introduced DNA or RNA has been “transformed” and is a“transformant” or a “clone.” The DNA or RNA introduced to a host cellcan come from any source, including cells of the same genus or speciesas the host cell, or cells of a different genus or species.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g. a foreign gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g. transcription and translation) of the introducedsequence. Vectors include plasmids, phages, viruses, etc.

Vectors typically comprise the DNA of a transmissible agent, into whichforeign DNA is inserted. A common way to insert one segment of DNA intoanother segment of DNA involves the use of enzymes called restrictionenzymes that cleave DNA at specific sites (specific groups ofnucleotides) called restriction sites. A “cassette” refers to a DNAcoding sequence or segment of DNA that codes for an expression productthat can be inserted into a vector at defined restriction sites. Thecassette restriction sites are designed to ensure insertion of thecassette in the proper reading frame. Generally, foreign DNA is insertedat one or more restriction sites of the vector DNA, and then is carriedby the vector into a host cell along with the transmissible vector DNA.A segment or sequence of DNA having inserted or added DNA, such as anexpression vector, can also be called a “DNA construct.” A common typeof vector is a “plasmid”, which generally is a self-contained moleculeof double-stranded DNA, usually of bacterial origin, that can readilyaccept additional (foreign) DNA and which can readily introduced into asuitable host cell. A plasmid vector often contains coding DNA andpromoter DNA and has one or more restriction sites suitable forinserting foreign DNA. Coding DNA is a DNA sequence that encodes aparticular amino acid sequence for a particular protein or enzyme.Promoter DNA is a DNA sequence which initiates, regulates, or otherwisemediates or controls the expression of the coding DNA. Promoter DNA andcoding DNA may be from the same gene or from different genes, and may befrom the same or different organisms. A large number of vectors,including plasmid and fungal vectors, have been described forreplication and/or expression in a variety of eukaryotic and prokaryotichosts. Non-limiting examples include pKK plasmids (Clonetech), pUCplasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREPplasmids (Invitrogen, San Diego, Calif.), or pMAL plasmids (New EnglandBiolabs, Beverly, Mass.), and many appropriate host cells, using methodsdisclosed or cited herein or otherwise known to those skilled in therelevant art. Recombinant cloning vectors will often include one or morereplication systems for cloning or expression, one or more markers forselection in the host, e.g. antibiotic resistance, and one or moreexpression cassettes.

The term “expression system” means a host cell and compatible vectorunder suitable conditions, e.g. for the expression of a protein codedfor by foreign DNA carried by the vector and introduced to the hostcell. Common expression systems include E. coli host cells and plasmidvectors, and insect host cells and Baculovirus vectors.

The term “heterologous” refers to a combination of elements notnaturally occurring together. For example, heterologous DNA refers toDNA not naturally located in the cell, or in a chromosomal site of thecell. Preferably, the heterologous DNA includes a gene foreign to thecell. A heterologous expression regulatory element is a such an elementoperatively associated with a different gene than the one it isoperatively associated with in nature. In the context of the presentinvention, an NRIF3 gene is heterologous to the vector DNA in which itis inserted for cloning or expression, and it is heterologous to a hostcell containing such a vector, in which it is expressed, e.g., a CHOcell.

The terms “mutant” and “mutation” mean any detectable change in geneticmaterial, e.g. DNA, or any process, mechanism, or result of such achange. This includes gene mutations, in which the structure (e.g. DNAsequence) of a gene is altered, any gene or DNA arising from anymutation process, and any expression product (e.g. protein or enzyme)expressed by a modified gene or DNA sequence. The term “variant” mayalso be used to indicate a modified or altered gene, DNA sequence,enzyme, cell, etc., i.e., any kind of mutant.

“Sequence-conservative variants” of a polynucleotide sequence are thosein which a change of one or more nucleotides in a given codon positionresults in no alteration in the amino acid encoded at that position.

“Function-conservative variants” are those in which a given amino acidresidue in a protein or enzyme has been changed without altering theoverall conformation and function of the polypeptide, including, but notlimited to, replacement of an amino acid with one having similarproperties (such as, for example, polarity, hydrogen bonding potential,acidic, basic, hydrophobic, aromatic, and the like). Amino acids withsimilar properties are well known in the art. For example, arginine,histidine and lysine are hydrophilic-basic amino acids and may beinterchangeable. Similarly, isoleucine, a hydrophobic amino acid, may bereplaced with leucine, methionine or valine. Such changes are expectedto have little or no effect on the apparent molecular weight orisoelectric point of the protein or polypeptide. Amino acids other thanthose indicated as conserved may differ in a protein or enzyme so thatthe percent protein or amino acid sequence similarity between any twoproteins of similar function may vary and may be, for example, from 70%to 99% as determined according to an alignment scheme such as by theCluster Method, wherein similarity is based on the MEGALIGN algorithm. A“function-conservative variant” also includes a polypeptide or enzymewhich has at least 60% amino acid identity as determined by BLAST orFASTA algorithms, preferably at least 75%, most preferably at least 85%,and even more preferably at least 90%, and which has the same orsubstantially similar properties or functions as the native or parentprotein or enzyme to which it is compared.

As used herein, the term “homologous” in all its grammatical forms andspelling variations refers to the relationship between proteins thatpossess a “common evolutionary origin,” including proteins fromsuperfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., Cell 50:667, 1987). Such proteins (and their encoding genes)have sequence homology, as reflected by their sequence similarity,whether in terms of percent similarity or the presence of specificresidues or motifs.

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that may or may not share a commonevolutionary origin (see Reeck et al., supra). However, in common usageand in the instant application, the term “homologous,” when modifiedwith an adverb such as “highly,” may refer to sequence similarity andmay or may not relate to a common evolutionary origin.

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 80%, and mostpreferably at least about 90 or 95%) of the nucleotides match over thedefined length of the DNA sequences, as determined by sequencecomparison algorithms, such as BLAST, FASTA, DNA Strider, etc. Anexample of such a sequence is an allelic or species variant of thespecific NRIF3 genes of the invention. Sequences that are substantiallyhomologous can be identified by comparing the sequences using standardsoftware available in sequence data banks, or in a Southernhybridization experiment under, for example, stringent conditions asdefined for that particular system.

Similarly, in a particular embodiment, two amino acid sequences are“substantially homologous” or “substantially similar” when greater than80% of the amino acids are identical, or greater than about 90% aresimilar (functionally identical). Preferably, the similar or homologoussequences are identified by alignment using, for example, the GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wis.) pileup program, or any of the programs described above(BLAST, FASTA, etc.).

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a T_(m)(melting temperature) of 55° C., can be used, e.g., 5×SSC, 0.1% SDS,0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS).Moderate stringency hybridization conditions correspond to a higherT_(m), e.g., 40% formamide, with 5× or 6×SCC. High stringencyhybridization conditions correspond to the highest T_(m), e.g., 50%formamide, 5× or 6×SCC. SCC is a 0.15M NaCl, 0.015M Na-citrate.Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of T_(m) for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherT_(m)) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating T_(m) have been derived (seeSambrook et al., supra, 9.50-9.51). For hybridization with shorternucleic acids, i.e., oligonucleotides, the position of mismatchesbecomes more important, and the length of the oligonucleotide determinesits specificity (see Sambrook et al., supra, 11.7-11.8). A minimumlength for a hybridizable nucleic acid is at least about 10 nucleotides;preferably at least about 15 nucleotides; and more preferably the lengthis at least about 20 nucleotides.

In a specific embodiment, the term “standard hybridization conditions”refers to a T_(m) of 55° C., and utilizes conditions as set forth above.In a preferred embodiment, the T_(m) is 60° C.; in a more preferredembodiment, the T_(m) is 65° C. In a specific embodiment, “highstringency” refers to hybridization and/or washing conditions at 68° C.in 0.2×SSC, at 42° C. in 50% formamide, 4×SSC, or under conditions thatafford levels of hybridization equivalent to those observed under eitherof these two conditions.

Another gene encoding NRIF3 (in addition to the full length cDNAdisclosed herein), whether genomic DNA or cDNA (such as splicevariants), can be isolated from any source, particularly from a humancDNA or genomic library. Methods for obtaining NRIF3 gene are well knownin the art, as described above (see, e.g., Sambrook et al., 1989,supra), The DNA may be obtained by standard procedures known in the artfrom cloned DNA (e.g., a DNA “library”), and preferably is obtained froma cDNA library prepared from tissues with high level expression of theprotein, by chemical synthesis, by cDNA cloning, or by the cloning ofgenomic DNA, or fragments thereof, purified from the desired cell (See,for example, Sambrook et al., 1989, supra; Glover, D. M. (ed.), 1985,DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I,II). Clones derived from genomic DNA may contain regulatory and intronDNA regions in addition to coding regions; clones derived from cDNA willnot contain intron sequences. Whatever the source, the gene should bemolecularly cloned into a suitable vector for propagation of the gene.Identification of the specific DNA fragment containing the desired NRIF3gene may be accomplished in a number of ways. For example, a portion ofa NRIF3 gene exemplified infra can be purified and labeled to prepare alabeled probe, and the generated DNA may be screened by nucleic acidhybridization to the labeled probe (Benton and Davis, Science 196:180,1977; Grunstein and Hogness, Proc. Natl. Acad. Sci. U.S.A. 72:3961,1975). Those DNA fragments with substantial homology to the probe, suchas an allelic variant from another individual, will hybridize. In aspecific embodiment, highest stringency hybridization conditions areused to identify a homologous NRIF3 gene.

Further selection can be carried out on the basis of the properties ofthe gene, e.g., if the gene encodes a protein product having theisoelectric, electrophoretic, amino acid composition, partial orcomplete amino acid sequence, antibody binding activity, or receptorbinding profile of NRIF3 protein as disclosed herein. Thus, the presenceof the gene may be detected by assays based on the physical, chemical,immunological, or functional properties of its expressed product.

The present invention also relates to cloning vectors containing genesencoding analogs and derivatives of NRIF3 of the invention. Theproduction and use of derivatives and analogs related to NRIF3 arewithin the scope of the present invention. For example, a deletionvariant form of functional NRIF3 can be provided. In a specificembodiment, the derivative or analog is functionally active, i.e.,capable of exhibiting one or more functional activities associated witha full-length, wild-type NRIF3 of the invention, or potentially blockingsuch function. Such functions include receptor binding activation orinhibition and localization to the cell nucleus. In another embodiment,an NRIF3 chimeric constructs fused with a non-NRIF3 protein are alsocontemplated. Examples of fusion partners include chimeric B42, GFPfusions, Gal4 fusion extension, etc.

NRIF3 derivatives can be made by altering encoding nucleic acidsequences by substitutions, additions or deletions that provide forfunctionally-active molecules. Preferably, derivatives are made thathave enhanced or increased functional activity relative to native NRIF3.Alternatively, such derivatives may encode dominant-negative fragmentsof NRIF3 that contain the receptor binding domain that have the same orgreater affinity for receptor.

Due to the degeneracy of nucleotide coding sequences, other DNAsequences which encode substantially the same amino acid sequence as aNRIF3 gene may be used in the practice of the present invention. Theseinclude but are not limited to allelic genes and nucleotide sequencescomprising all or portions of NRIF3 genes which are altered by thesubstitution of different codons that encode the same amino acid residuewithin the sequence, thus producing a silent change. Likewise, the NRIF3derivatives of the invention include, but are not limited to, thosecontaining, as a primary amino acid sequence, all or part of the aminoacid sequence of a NRIF3 protein including altered sequences in whichfunctionally equivalent amino acid residues are substituted for residueswithin the sequence resulting in a conservative amino acid substitution.For example, one or more amino acid residues within the sequence can besubstituted by another amino acid of a similar polarity and, if present,charge, which acts as a functional equivalent, resulting in a silentalteration. Substitutes for an amino acid within the sequence may beselected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine. Amino acids containing aromatic ring structures arephenylalanine, tryptophan, and tyrosine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Such alterations will not beexpected to affect apparent molecular weight as determined bypolyacrylamide gel electrophoresis, or isoelectric point. Particularlypreferred substitutions are:

-   -   Lys for Arg and vice versa such that a positive charge may be        maintained;    -   Glu for Asp and vice versa such that a negative charge may be        maintained;    -   Ser for Thr such that a free —OH can be maintained; and    -   Gln for Asn such that a free CONH₂ can be maintained.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys.

The genes encoding NRIF3 derivatives and analogs of the invention can beproduced by various methods known in the art. The manipulations whichresult in their production can occur at the gene or protein level. Forexample, the cloned NRIF3 gene sequence can be modified by any ofnumerous strategies known in the art (Sambrook et al., 1989, supra). Thesequence can be cleaved at appropriate sites with restrictionendonuclease(s), followed by further enzymatic modification if desired,isolated, and ligated in vitro. In the production of the gene encoding aderivative or analog of NRIF3, care should be taken to ensure that themodified gene remains within the same translational reading frame as theNRIF3 gene, uninterrupted by translational stop signals, in the generegion where the desired activity is encoded.

Additionally, the NRIF3-encoding nucleic acid sequence can be mutated invitro or in vivo, to create and/or destroy translation, initiation,and/or termination sequences, or to create variations in coding regionsand/or form new restriction endonuclease sites or destroy preexistingones, to facilitate further in vitro modification. In the Examples,infra, such modifications were made to introduce restriction sites andfacilitate cloning the NRIF3 gene into an expression vector. Anytechnique for mutagenesis known in the art can be used, including butnot limited to, in vitro site-directed mutagenesis (Hutchinson, C., etal., J. Biol. Chem. 253:6551, 1978; Zoller and Smith, DNA 3:479-488,1984; Oliphant et al., Gene 44:177, 1986; Hutchinson et al., Proc. Natl.Acad. Sci. U.S.A. 83:710, 1986), use of TAB linkers (Pharmacia), etc.PCR techniques are preferred for site directed mutagenesis (see Higuchi,1989, “Using PCR to Engineer DNA”, in PCR Technology: Principles andApplications for DNA Amplification, H. Erlich, ed., Stockton Press,Chapter 6, pp. 61-70).

The identified and isolated gene can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Examples of vectors include, but arenot limited to, E. coli, bacteriophages such as lambda derivatives, orplasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g.,pGEX vectors, pmal-c, pFLAG, etc. The insertion into a cloning vectorcan, for example, be accomplished by ligating the DNA fragment into acloning vector which has complementary cohesive termini. However, if thecomplementary restriction sites used to fragment the DNA are not presentin the cloning vector, the ends of the DNA molecules may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers may comprise specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences.

Recombinant molecules can be introduced into host cells viatransformation, transfection, infection, electroporation, etc., so thatmany copies of the gene sequence are generated. Preferably, the clonedgene is contained on a shuttle vector plasmid, which provides forexpansion in a cloning cell, e.g., E. coli, and facile purification forsubsequent insertion into an appropriate expression cell line, if suchis desired. For example, a shuttle vector, which is a vector that canreplicate in more than one type of organism, can be prepared forreplication in both E. coli and Saccharomyces cerevisiae by linkingsequences from an E. coli plasmid with sequences from the yeast 2μplasmid.

Expression of NRIF3 Polypeptides

The nucleotide sequence coding for NRIF3, or antigenic fragment,derivative or analog thereof, or a functionally active derivative,including a chimeric protein, thereof, can be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequence. Thus, the nucleic acid encoding NRIF3 of theinvention is operationally associated with a promoter in an expressionvector of the invention. Both cDNA and genomic sequences can be clonedand expressed under control of such regulatory sequences. An expressionvector also preferably includes a replication origin.

Alternatively, an NRIF3 polypeptide of the invention can be preparedusing well-known techniques in peptide synthesis, including solid phasesynthesis (using, e.g., BOC of FMOC chemistry), or peptide condensationtechniques.

As used herein, the terms “polypeptide” and “protein” may be usedinterchangeably to refer to the gene product (or corresponding syntheticproduct) of an NRIF3 gene. The term “protein” may also referspecifically to the polypeptide as expressed in cells. A peptide isgenerally a fragment of a polypeptide, e.g., of about six or more aminoacid residues.

The necessary transcriptional and translational signals can be providedon a recombinant expression vector, or they may be supplied by thenative gene encoding NRIF3 and/or its flanking regions.

Potential host-vector systems include but are not limited to mammaliancell systems infected with virus (e.g., vaccinia virus, adenovirus,adeno-associated virus, herpes virus, etc.); insect cell systemsinfected with virus (e.g., baculovirus); microorganisms such as yeastcontaining yeast vectors; or bacteria transformed with bacteriophage,DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors varyin their strengths and specificities. Depending on the host-vectorsystem utilized, any one of a number of suitable transcription andtranslation elements may be used. A preferred expression host is aeukaryotic cell (e.g., yeast, insect, or mammalian cell). More preferredis a mammalian cell, e.g., human, rat, monkey, dog, or hamster cell.

A recombinant NRIF3 protein of the invention, or functional fragment,derivative, chimeric construct, or analog thereof, may be expressedchromosomally, after integration of the coding sequence byrecombination. In this regard, any of a number of amplification systemsmay be used to achieve high levels of stable gene expression (SeeSambrook et al., 1989, supra).

Any of the methods previously described for the insertion of DNAfragments into a cloning vector may be used to construct expressionvectors containing a gene consisting of appropriatetranscriptional/translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombination (genetic recombination).

Expression of NRIF3 protein may be controlled by any promoter/enhancerelement known in the art, but these regulatory elements must befunctional in the host selected for expression. Promoters which may beused to control NRIF3 gene expression include, but are not limited to,cytomegalovirus (CMV) promoter (U.S. Pat. Nos. 5,385,839 and 5,168,062),the SV40 early promoter region (Benoist and Chambon, 1981, Nature290:304-310), the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto, et al., Cell 22:787-797, 1980), the herpesthymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A.78:1441-1445, 1981), the regulatory sequences of the metallothioneingene (Brinster et al., Nature 296:39-42, 1982); prokaryotic expressionvectors such as the β-lactamase promoter (Villa-Kamaroff, et al., Proc.Natl. Acad. Sci. U.S.A. 75:3727-3731, 1978), or the tac promoter(DeBoer, et al., Proc. Natl. Acad. Sci. U.S.A. 80:21-25, 1983); see also“Useful proteins from recombinant bacteria” in Scientific American,242:74-94, 1980; promoter elements from yeast or other fungi such as theGal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK(phosphoglycerol kinase) promoter, alkaline phosphatase promoter; andthe animal transcriptional control regions, which exhibit tissuespecificity and have been utilized in transgenic animals: elastase Igene control region which is active in pancreatic acinar cells (Swift etal., Cell 38:639-646, 1984; Ornitz et al., Cold Spring Harbor Symp.Quant. Biol. 50:399-409, 1986; MacDonald, Hepatology 7:425-515, 1987);insulin gene control region which is active in pancreatic beta cells(Hanahan, Nature 315:115-122, 1985), immunoglobulin gene control regionwhich is active in lymphoid cells (Grosschedl et al., Cell 38:647-658,1984; Adames et al., Nature 318:533-538, 1985; Alexander et al., Mol.Cell. Biol. 7:1436-1444, 1987), mouse mammary tumor virus control regionwhich is active in testicular, breast, lymphoid and mast cells (Leder etal., Cell 45:485-495, 1986), albumin gene control region which is activein liver (Pinkert et al., Genes and Devel. 1:268-276, 1987),alpha-fetoprotein gene control region which is active in liver (Krumlaufet al., Mol. Cell. Biol. 5:1639-1648, 1985; Hammer et al., Science235:53-58, 1987), alpha 1-antitrypsin gene control region which isactive in the liver (Kelsey et al., Genes and Devel. 1: 161-171, 1987),beta-globin gene control region which is active in myeloid cells (Mogramet al., Nature 315:338-340, 1985; Kollias et al., Cell 46:89-94, 1986),myelin basic protein gene control region which is active inoligodendrocyte cells in the brain (Readhead et al., Cell 48:703-712,1987), myosin light chain-2 gene control region which is active inskeletal muscle (Sani, Nature 314:283-286, 1985), and gonadotropicreleasing hormone gene control region which is active in thehypothalamus (Mason et al., Science 234:1372-1378, 1986).

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol E1, pCR1, pBR322, pMal-C2, pET, pGEX (Smith et al., Gene 67:31-40,1988), pMB9 and their derivatives, plasmids such as RP4; phage DNAs,e.g., the numerous derivatives of phage 1, e.g., NM989, and other phageDNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmidssuch as the 2μ plasmid or derivatives thereof, vectors useful ineukaryotic cells, such as vectors useful in insect or mammalian cells;vectors derived from combinations of plasmids and phage DNAs, such asplasmids that have been modified to employ phage DNA or other expressioncontrol sequences; and the like.

Yeast expression systems can also be used according to the invention toexpress NRIF3. For example, the non-fusion pYES2 vector (XbaI, SphI,ShoI, NotI, GstXI, EcoRI, BstXI, BamH1, SacI, Kpn1, and HindIII cloningsites; Invitrogen) or the fusion pYESHisA, B, C (XbaI, SphI, ShoI, NotI,BstXI, EcoRI, BamH1, SacI, KpnI, and HindIII cloning sites, N-terminalpeptide purified with ProBond resin and cleaved with enterokinase;Invitrogen), to mention just two, can be employed according to theinvention. As exemplified infra, a yeast two-hybrid expression systemcan be prepared in accordance with the invention.

Vectors are introduced into the desired host cells by methods known inthe art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al., J. Biol. Chem.267:963-967, 1992; Wu and Wu, J. Biol. Chem. 263:14621-14624, 1988;Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar.15, 1990).

Antibodies to NRIF3

According to the invention, NRIF3 polypeptides produced recombinantly orby chemical synthesis, and fragments or other derivatives or analogsthereof, including fusion proteins, may be used as an immunogen togenerate antibodies that recognize the NRIF3 polypeptide. Suchantibodies include but are not limited to polyclonal, monoclonal,chimeric, single chain, Fab fragments, and an Fab expression library.Such an antibody is specific for human NRIF3.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to NRIF3 polypeptide or derivative or analogthereof. For the production of antibody, various host animals can beimmunized by injection with the NRIF3 polypeptide, or a derivative(e.g., fragment or fusion protein) thereof, including but not limited torabbits, mice, rats, sheep, goats, etc. In one embodiment, the NRIF3polypeptide or fragment thereof can be conjugated to an immunogeniccarrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH). Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward the NRIF3polypeptide, or fragment, analog, or derivative thereof, any techniquethat provides for the production of antibody molecules by continuouscell lines in culture may be used. These include but are not limited tothe hybridoma technique originally developed by Kohler and Milstein(Nature 256:495-497, 1975), as well as the trioma technique, the humanB-cell hybridoma technique (Kozbor et al., Immunology Today 4:72, 1983;Cote et al, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030, 1983), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole etal., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96, 1985). Production of human antibodies by CDR grafting isdescribed in U.S. Pat. Nos. 5,585,089, 5,693,761, and 5,693,762 to Queenet al., and also in U.S. Pat. No. 5,225,539 to Winter and InternationalPatent Application PCT/WO91/09967 by Adau et al. In an additionalembodiment of the invention, monoclonal antibodies can be produced ingerm-free animals (International Patent Publication No. WO 89/12690,published 28 Dec. 1989). In fact, according to the invention, techniquesdeveloped for the production of “chimeric antibodies” (Morrison et al.,J. Bacteriol. 159:870, 1984); Neuberger et al., Nature 312:604-608,1984; Takeda et al., Nature 314:452-454, 1985) by splicing the genesfrom a mouse antibody molecule specific for an NRIF3 polypeptidetogether with genes from a human antibody molecule of appropriatebiological activity can be used; such antibodies are within the scope ofthis invention. Such human or humanized chimeric antibodies arepreferred for use in therapy of human diseases or disorders (describedinfra), since the human or humanized antibodies are much less likelythan xenogenic antibodies to induce an immune response, in particular anallergic response, themselves.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. Nos. 5,476,786 and 5,132,405 toHuston; U.S. Pat. No. 4,946,778) can be adapted to produce NRIF3polypeptide-specific single chain antibodies. An additional embodimentof the invention utilizes the techniques described for the constructionof Fab expression libraries (Huse et al., Science 246:1275-1281, 1989)to allow rapid and easy identification of monoclonal Fab fragments withthe desired specificity for an NRIF3 polypeptide, or its derivatives, oranalogs.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of an NRIF3 polypeptide, one may assay generatedhybridomas for a product which binds to an NRIF3 polypeptide fragmentcontaining such epitope. For selection of an antibody specific to anNRIF3 polypeptide from a particular species of animal, one can select onthe basis of positive binding with NRIF3 polypeptide expressed by orisolated from cells of that species of animal.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the NRIF3 polypeptide,e.g., for Western blotting, imaging NRIF3 polypeptide in situ, measuringlevels thereof in appropriate physiological samples, etc. using any ofthe detection techniques mentioned above or known in the art. Suchantibodies can be used to identify proteins that interact with NRIF3.

In a specific embodiment, antibodies that agonize or antagonize theactivity of NRIF3 polypeptide can be generated. They can also be used toregulate or inhibit NRIF3 activity intracellular, i.e., the inventioncontemplates an intracellular antibody (intrabody), e.g., single chainFv antibodies (see generally, Chen, Mol. Med. Today, 3:160-167, 1997;Spitz et al., Anticancer Res., 16:3415-3422, 1996; Indolfi et al., Nat.Med., 2:634-635, 1996; Kijima et al., Pharmacol. Ther., 68:247-267,1995).

Screening and Chemistry

Identification and isolation of NRIF3 provides for development ofscreening assays, particularly for high throughput screening ofmolecules that up- or down-regulate the activity of NRIF3, e.g., bypermitting expression of NRIF3 in quantities greater than can beisolated from natural sources, or in indicator cells that are speciallyengineered to indicate the activity of NRIF3 expressed aftertransfection or transformation of the cells. In addition, the presentinvention contemplates methods for identifying specific ligands ofthyroid hormone receptor or retinoid X receptor using various screeningassays known in the art.

Any screening technique known in the art can be used to screen for NRIF3agonists or antagonists. The present invention contemplates screens forsmall molecule ligands or ligand analogs and mimics, as well as screensfor natural ligands that bind to and agonize or antagonize the activityof NRIF3 in vivo. For example, natural products libraries can bescreened using assays of the invention for molecules that agonize orantagonize NRIF3 activity.

As used herein, the term “ligand” refers to compounds.

Knowledge of the primary sequence of the protein, and the similarity ofthat sequence with proteins of known function, can provide an initialclue as to the inhibitors or antagonists of the protein. Identificationand screening of antagonists is further facilitated by determiningstructural features of the protein, e.g., using X-ray crystallography,neutron diffraction, nuclear magnetic resonance spectrometry, and othertechniques for structure determination. These techniques provide for therational design or identification of agonists and antagonists.

Another approach uses recombinant bacteriophage to produce largelibraries. Using the “phage method” (Scott and Smith, Science249:386-390, 1990; Cwirla, et al., Proc. Natl. Acad. Sci., 87:6378-6382,1990; Devlin et al., Science, 49:404-406, 1990), very large librariescan be constructed (10⁶-10⁸ chemical entities). A second approach usesprimarily chemical methods, of which the Geysen method (Geysen et al.,Molecular Immunology 23:709-715, 1986; Geysen et al. J. ImmunologicMethod 102:259-274, 1987; and the method of Fodor et al. (Science251:767-773, 1991) are examples. Furka et al. (14th InternationalCongress of Biochemistry, Volume #5, Abstract FR:013, 1988; Furka, Int.J. Peptide Protein Res. 37:487-493, 1991), Houghton (U.S. Pat. No.4,631,211, issued December 1986) and Rutter et al. (U.S. Pat. No.5,010,175, issued Apr. 23, 1991) describe methods to produce a mixtureof peptides that can be tested as agonists or antagonists.

In another aspect, synthetic libraries (Needels et al., Proc. Natl.Acad. Sci. USA 90:10700-4, 1993; Ohlmeyer et al., Proc. Natl. Acad. Sci.USA 90:10922-10926, 1993; Lam et al., International Patent PublicationNo. WO 92/00252; Kocis et al., International Patent Publication No. WO9428028) and the like can be used to screen for NRIF3 ligands accordingto the present invention.

Screening System for TR and RXR Agonists and Antagonists

In a specific embodiment, in addition to directly screening formolecules that directly interact with NRIF3, such as antibodies or smallmolecules, which can serve as negative regulators (or even positiveregulators) of NRIF3 activity, the present invention provides thecomponents to reconstitute thyroid hormone receptor and retinoid-Xreceptor signaling activity in whole or in part. This reconstitutedsystem, which depends on recombinant expression of TR and RXR with NRIF3in a host cell, permits discovery and evaluation of agonists andantagonists of receptor signally (a signal-inducing ligand is anagonist; a non-signal inducing ligand is an antagonist). Such agonistsand antagonist can act at the level of TR or RXR binding, i.e., asreceptor ligands, or at the level of signal transduction. Modulation ofsignal transduction can occur by modulating (inhibiting or promoting)receptor-coactivator (NRIF3) interaction, or NRIF3 activity, throughbinding to NRIF3 and/or receptor.

Any host cell that provides for expression of functional TR or RXR andNRIF3 proteins can be used, e.g., a eukaryotic cell. In a specificembodiment, the yeast system described in the examples can be modifiedfor use in a screening assay of this sort. Alternatively, one can employa mammalian or insect cells genetically engineered to express the TR orRXR and NRIF3 proteins. In a specific embodiment, a host cell harbors aconstruct that expresses a nuclear hormone receptor (TR or RXR or itsligand binding domain) fused to a DNA binding domain and anotherconstruct expressing NIRF3 fused to an activation domain. Alternatively,the host cell harbors a construct expressing NRIF3 fused to a DNAbinding domain and another construct expressing a receptor (TR or RXR ora ligand-binding domain thereof) fused to an activation domain. The hostcell will also include a reporter gene that is expressed in response tobinding of the nuclear hormone receptor-NRIF3 complex (formed as aresult of binding of ligand to the nuclear hormone receptor) to anexpression control sequence operatively associated with the reportergene. Reporter genes for use in the invention encode detectableproteins, including, but by no means limited to, chloramphenicoltransferase (CAT), β-galactosidase (β-gal), luciferase, greenfluorescent protein (GFP), alkaline phosphatase, and other genes thatcan be detected, e.g., immunologically (by antibody assay). GFP has beenmodified to produce proteins that remain functional but have differentfluorescent properties. Heim et al (U.S. Pat. No. 5,625,048) modifiedGFP resulting in amino-acid changes which exhibited different excitationand emission spectra with visibly distinct colors and increasedintensities of emission. Bjorn et al (WO 9623898) developed a newconstruct which encoded a modified GFP but also contained an enzymerecognition site. Bjorn et al (WO9711094) also developed new fluorescentproteins with increased intensity compared to the parent proteins.Hauswirth et al (WO97266333) developed a GFP protein optimized toprovide higher levels of expression in mammalian cells. Gaitanaris et al(WO9742320) modified GFP resulting to increase the intensity offluorescence, e.g., by some twenty times greater than wild-type GFP,therefore increasing the sensitivity of detection. Cubitt et al(WO9806737) developed modified GFP which could be easily distinguishedfrom the already known green and blue fluorescent proteins. Evans et al(WO9821355) developed new GFP mutants excitable with blue and whitelight.

The host cell screening system of the invention permits two kinds ofassays: direct activation assays (agonist screen) and inhibition assays(antagonist screen). An agonist screen involves detecting expression ofthe reporter gene by the host cell contacted with a test compound. Ifthe reporter gene is expressed, the test compound has inducedassociation of the nuclear receptor and NRIF3, and the test compound isa candidate agonist of the nuclear receptor signal. If there is no orvery low expression of the reporter gene, no such association and geneactivation has occurred, and the test compound is not an effectiveagonist.

An antagonist screen involves detecting expression of the reporter geneby the host cell when contacted with the nuclear hormone receptor ligand(or another agonist) and a test compound. If reporter gene expression isreduced or eliminated, the test compound has prevented activation ofgene expression, which may occur by competitively or non-competitivelyinhibiting binding of the ligand (or agonist) to the nuclear hormonereceptor; preventing association of the receptor and NRIF3. Such a testcompound is a candidate antagonist of nuclear hormone receptorsignaling. If there is no change in expression of the reporter gene, thetest compound is not an effective antagonist.

The reporter gene assay system described here may be used in ahigh-throughput primary screen for agonists and antagonists, or it maybe used as a secondary functional screen for candidate compoundsidentified by a different primary screen, e.g., a binding assay screenthat identifies compounds that interact with the receptor and/or NRIF3.

Modulation of NRIF3 Activity

Since NRIF3 is a co-activator of the thyroid hormone receptors (TRs) andthe retinoid X receptors (RXRs), a defect in the NRIF3 function (e.g.,caused by genetic mutations) can result in defects in one or both ofthese two receptor-signalling pathways. For example, a defect in NRIF3may lead to a weakened response to thyroid hormones and, therefore,results in functional hypothyroidism. In addition to their roles in theretinoid signalling, the RXRs are involved in the function of many othernuclear receptors through heterodimerization with these receptors. In anumber of such heterodimers, the RXR partner is capable of binding itsligand and, therefore, regulating the expression of the target genes inconjunction with the other receptor. Therefore, a defect in the NRIF3may (i) affect the retinoid signalling and lead to an abnormal responseto vitamin A; or (ii) affect other physiological processes that are(partially) regulated by RXRs through the heterodimerization with otherreceptors. An example of such a heterodimer is PPARγ/RXR, which has beenan important target for developing drugs for the treatment of type IIdiabetes. It has been shown in clinical and in experimental modelsystems that ligands for both PPARγ and RXR can sensitize the responseto insulin and, therefore, have therapeutic values for type II diabetes,presumably through up-regulation of target genes that are important inmediating insulin signalling. It is an interesting possibility that adefect in NRIF3 may weaken the signalling of the PPARγ/RXR heterodimerin vivo and may contribute to the development of type II diabetes andlead to weakened response to drugs that target the PPARγ/RXRheterodimer.

In the case when a defect in NRIF3 causes the disease phenotype (such asthe diseases discussed in the above paragraph), one of the therapeuticoptions would be to correct such a defect through the introduction of afunctional NRIF3 (e.g., by using a NRIF3 gene therapy). As discussedabove, such diseases may include functional hypothyroidism, abnormalresponse to vitamin A, and type II diabetes.

On the other hand, even if the endogenous NRIF3 is not defective and thedisease is not directly related to NRIF3, NRIF3 still be used in thetreatment of such a disease if up-regulation of the correspondinghormone signalling pathways would be beneficial. For example, even whenthe hypothyroidism or type II diabetes are not caused by a defect inNRIF3, additional expression of NRIF3 would enhance the correspondinghormone signalling pathways and, therefore, could be used to treat thediseases (either alone or in conjunction with other drugs that alsoup-regulate the targeted hormone pathways).

Retinoids have been shown to inhibit the growth and/or inducedifferentiation of a number of cancer cells. Retinoid signalling ismediated by the RAR/RXR heterodimer. When the RAR partner is occupied byits ligand, the RXR becomes a permissive partner and can engage in itsown ligand binding and subsequent signalling. In such a case it ispossible that the additional introduction of NRIF3 would enhance thesignal output from an activated RAR/RXR heterodimer and thus potentiatethe effect of the retinoids in inhibiting the growth of cancer cellsand/or in inducing their differentiation. In addition, certain breastcancer cells are resistant to RAR agonists. However, treatment withRXR-specific ligands can inhibit the growth of these cells and/or inducetheir sensitivities to RAR agonists. In such a case, the introduction ofNRIF3 may enhance the anti-cancer effect of RXR-specific ligands. Insummary, NRIF3 can be used in treating cancers in conjunction withretinoids and/or rexinoids.

RXR-specific ligands have also been shown to be efficientchemoprevention agents for cancers and type II diabetes in experimentalmodels systems. The introduction of NRIF3 would further enhance theeffects of such RXR ligands in preventing the onset of these diseases.Finally, since RXRs are also involved in many other receptor-signallingpathways, NRIF3 may have therapeutic potentials in diseases related tosuch receptors as well.

In the case of a disease that is caused by “over-signalling” (e.g.,hyperthyroidism) of a certain hormone pathway that involves NRIF3, itwill be of therapeutic benefit to down-regulate the pathway by targetingendogenous NRIF3. There are several different ways to target NRIF3: (i)through the introduction of antisense oligonucleotides against NRIF3;(ii) through suitable modification of NRIF3 to create a dominantnegative construct (if applicable); (iii) through neutralizinganti-NRIF3 intrabodies; and (iv) through small molecules that mimicNRIF3 in its binding to the liganded receptor, but do not relay theactivation signal(s).

In Vivo Testing Using Transgenic Animals

Transgenic mammals can be prepared for evaluating the molecularmechanisms of NRIF3, and particularly human NRIF3/TR- orNRIF3/RXR-induced signaling. Such mammals provide excellent models forscreening or testing drug candidates. It is possible to evaluatecompounds or diseases on “knockout” animals, e.g., to identify acompound that can compensate for a defect in NRIF3 activity.Alternatively, human NRIF3 or TR/RXR, or both (double transgenics),“knock-in” mammals can be prepared for evaluating the molecular biologyof this system in greater detail than is possible with human subjects(although the close evolutionary relationship of TR and RXR likelyobviate the need to use the human forms of these receptors. Bothtechnologies permit manipulation of single units of genetic informationin their natural position in a cell genome and to examine the results ofthat manipulation in the background of a terminally differentiatedorganism. These animals can be evaluated for hyperthyroidism,hypothyroidism, or susceptibility to diabetes cancer, e.g., in responseto challenge of carcinogens, by increasing or decreasing RXR response).

A “knockout mammal” is a mammal (e.g., mouse) that contains within itsgenome a specific gene that has been inactivated by the method of genetargeting (see, e.g., U.S. Pat. Nos. 5,777,195 and 5,616,491). Aknockout mammal includes both a heterozygote knockout (i.e., onedefective allele and one wild-type allele) and a homozygous mutant.Preparation of a knockout mammal requires first introducing a nucleicacid construct that will be used to suppress expression of a particulargene into an undifferentiated cell type termed an embryonic stem cell.This cell is then injected into a mammalian embryo. A mammalian embryowith an integrated cell is then implanted into a foster mother for theduration of gestation. Zhou, et al. (Genes and Development, 9:2623-34,1995) describes PPCA knock-out mice. Knockout mice can be used to studyhypothyroidism, vitamin A responses, type II diabetes, and cancersusceptibility. Disease phenotypes that develop can provide a platformfor further drug discovery.

The term “knockout” refers to partial or complete suppression of theexpression of at least a portion of a protein encoded by an endogenousDNA sequence in a cell. The term “knockout construct” refers to anucleic acid sequence that is designed to decrease or suppressexpression of a protein encoded by endogenous DNA sequences in a cell.The nucleic acid sequence used as the knockout construct is typicallycomprised of (1) DNA from some portion of the gene (exon sequence,intron sequence, and/or promoter sequence) to be suppressed and (2) amarker sequence used to detect the presence of the knockout construct inthe cell. The knockout construct is inserted into a cell, and integrateswith the genomic DNA of the cell in such a position so as to prevent orinterrupt transcription of the native DNA sequence. Such insertionusually occurs by homologous recombination (i.e., regions of theknockout construct that are homologous to endogenous DNA sequenceshybridize to each other when the knockout construct is inserted into thecell and recombine so that the knockout construct is incorporated intothe corresponding position of the endogenous DNA). The knockoutconstruct nucleic acid sequence may comprise 1) a full or partialsequence of one or more exons and/or introns of the gene to besuppressed, 2) a full or partial promoter sequence of the gene to besuppressed, or 3) combinations thereof. Typically, the knockoutconstruct is inserted into an embryonic stem cell (ES cell) and isintegrated into the ES cell genomic DNA, usually by the process ofhomologous recombination. This ES cell is then injected into, andintegrates with, the developing embryo.

The phrases “disruption of the gene” and “gene disruption” refer toinsertion of a nucleic acid sequence into one region of the native DNAsequence (usually one or more exons) and/or the promoter region of agene so as to decrease or prevent expression of that gene in the cell ascompared to the wild-type or naturally occurring sequence of the gene.By way of example, a nucleic acid construct can be prepared containing aDNA sequence encoding an antibiotic resistance gene which is insertedinto the DNA sequence that is complementary to the DNA sequence(promoter and/or coding region) to be disrupted. When this nucleic acidconstruct is then transfected into a cell, the construct will integrateinto the genomic DNA. Thus, many progeny of the cell will no longerexpress the gene at least in some cells, or will express it at adecreased level, as the DNA is now disrupted by the antibioticresistance gene.

A “knock-in” mammal is a mammal in which an endogenous gene issubstituted with a heterologous gene (Roemer et al., New Biol. 3:331,1991). Preferably, the heterologous gene is “knocked-in” to a locus ofinterest, either the subject of evaluation (in which case the gene maybe a reporter gene; see Elefanty et al., Proc Natl Acad Sci USA95:11897, 1998) of expression or function of a homologous gene, therebylinking the heterologous gene expression to transcription from theappropriate promoter. This can be achieved by homologous recombination,transposon (Westphal and Leder, Curr Biol 7:530, 1997), using mutantrecombination sites (Araki et al., Nucleic Acids Res 25:868, 1997) orPCR (Zhang and Henderson, Biotechniques 25:784, 1998).

Generally, for homologous recombination, the DNA will be at least about1 kilobase (kb) in length and preferably 3-4 kb in length, therebyproviding sufficient complementary sequence for recombination when theknockout construct is introduced into the genomic DNA of the ES cell(discussed below).

Included within the scope of this invention is a mammal in which two ormore genes have been knocked out or knocked in, or both. Such mammalscan be generated by repeating the procedures set forth herein forgenerating each knockout construct, or by breeding to mammals, each witha single gene knocked out, to each other, and screening for those withthe double knockout genotype.

Regulated knockout animals can be prepared using various systems, suchas the tet-repressor system (see U.S. Pat. No. 5,654,168) or the Cre-Loxsystem (see U.S. Pat. Nos. 4,959,317 and 5,801,030).

In another series of embodiments, transgenic animals are created inwhich (i) a human NRIF3 is stably inserted into the genome of thetransgenic animal; and/or (ii) the endogenous NRIF3 genes areinactivated and replaced with their human counterparts. See, e.g.,Coffman, Semin. Nephrol. 17:404, 1997; Esther et al., Lab. Invest.74:953, 1996; Murakami et al., Blood Press. Suppl. 2:36, 1996. Suchanimals can be treated with candidate compounds and monitored for theeffects of such drugs on NRIF3 activity.

Gene Therapy to Modulate NRIF3 Activity

A gene encoding NRIF3, or alternatively a negative regulator of NRIF3such as an antisense nucleic acid, intracellular antibody (intrabody),or dominant negative NIRF3 (which may be truncated), can be introducedin vivo, ex vivo, or in vitro using a viral or a non-viral vector, e.g.,as discussed above. Expression in targeted tissues can be effected bytargeting the transgenic vector to specific cells, such as with a viralvector or a receptor ligand, or by using a tissue-specific promoter, orboth. Targeted gene delivery is described in International PatentPublication WO 95/28494, published October 1995.

Preferably, for in vivo administration, an appropriate immunosuppressivetreatment is employed in conjunction with the viral vector, e.g.,adenovirus vector, to avoid immuno-deactivation of the viral vector andtransfected cells. For example, immunosuppressive cytokines, such asinterleukin-12 (IL-12), interferon-γ (IFN-γ), or anti-CD4 antibody, canbe administered to block humoral or cellular immune responses to theviral vectors (see, e.g., Wilson, Nature Medicine, 1995). In thatregard, it is advantageous to employ a viral vector that is engineeredto express a minimal number of antigens.

Adenovirus vectors. Adenoviruses are eukaryotic DNA viruses that can bemodified to efficiently deliver a nucleic acid of the invention to avariety of cell types in vivo, and has been used extensively in genetherapy protocols. Various serotypes of adenovirus exist. Of theseserotypes, preference is given to using type 2 or type 5 humanadenoviruses (Ad 2 or Ad 5) or adenoviruses of animal origin (seeWO94/26914). Those adenoviruses of animal origin which can be usedwithin the scope of the present invention include adenoviruses ofcanine, bovine, murine (example: Mav1, Beard et al., Virology 75 (1990)81), ovine, porcine, avian, and simian (example: SAV) origin.Preferably, the adenovirus of animal origin is a canine adenovirus, morepreferably a CAV2 adenovirus (e.g., Manhattan or A26/61 strain (ATCCVR-800), for example). Various replication defective adenovirus andminimum adenovirus vectors have been described for gene therapy(WO94/26914, WO95/02697, WO94/28938, WO94/28152, WO94/12649, WO95/02697WO96/22378). The replication defective recombinant adenovirusesaccording to the invention can be prepared by any technique known to theperson skilled in the art (Levrero et al., Gene 101:195 1991; EP 185573; Graham, EMBO J. 3:2917, 1984; Graham et al., J. Gen. Virol. 36:591977). Recombinant adenoviruses are recovered and purified usingstandard molecular biological techniques, which are well known to one ofordinary skill in the art.

Adeno-associated viruses. The adeno-associated viruses (AAV) are DNAviruses of relatively small size which can integrate, in a stable andsite-specific manner, into the genome of the cells which they infect.They are able to infect a wide spectrum of cells without inducing anyeffects on cellular growth, morphology or differentiation, and they donot appear to be involved in human pathologies. The AAV genome has beencloned, sequenced and characterized. The use of vectors derived from theAAVs for transferring genes in vitro and in vivo has been described (seeWO 91/18088; WO 93/09239; U.S. Pat. Nos. 4,797,368, 5,139,941, EP 488528). The replication defective recombinant AAVs according to theinvention can be prepared by co-transfecting a plasmid containing thenucleic acid sequence of interest flanked by two AAV inverted terminalrepeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes(rep and cap genes), into a cell line which is infected with a humanhelper virus (for example an adenovirus). The AAV recombinants which areproduced are then purified by standard techniques.

Retrovirus vectors. In another embodiment the gene can be introduced ina retroviral vector, e.g., as described in Anderson et al., U.S. Pat.No. 5,399,346; Mann et al., 1983, Cell 33:153; Temin et al., U.S. Pat.No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al.,1988, J. Virol. 62:1120; Temin et al., U.S. Pat. No. 5,124,263; EP453242, EP178220; Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick,BioTechnology 3 (1985) 689; International Patent Publication No. WO95/07358, published Mar. 16, 1995, by Dougherty et al.; and Kuo et al.,1993, Blood 82:845. The retroviruses are integrating viruses whichinfect dividing cells. The retrovirus genome includes two LTRs, anencapsidation sequence and three coding regions (gag, pol and env). Inrecombinant retroviral vectors, the gag, pol and env genes are generallydeleted, in whole or in part, and replaced with a heterologous nucleicacid sequence of interest. These vectors can be constructed fromdifferent types of retrovirus, such as MoMuLV (“murine Moloney leukaemiavirus”), MSV (“murine Moloney sarcoma virus”), HaSV (“Harvey sarcomavirus”); SNV (“spleen necrosis virus”); RSV (“Rous sarcoma virus”) andFriend virus. Suitable packaging cell lines have been described in theprior art, in particular the cell line PA317 (U.S. Pat. No. 4,861,719);the PsiCRIP cell line (WO 90/02806) and the GP+envAm-12 cell line (WO89/07150). In addition, the recombinant retroviral vectors can containmodifications within the LTRs for suppressing transcriptional activityas well as extensive encapsidation sequences which may include a part ofthe gag gene (Bender et al, J. Virol. 61:1639, 1987). Recombinantretroviral vectors are purified by standard techniques known to thosehaving ordinary skill in the art.

Retrovirus vectors can also be introduced by recombinant DNA viruses,which permits one cycle of retroviral replication and amplifiestransfection efficiency (see WO 95/22617, WO 95/26411, WO 96/39036, WO97/19182).

Lentivirus vectors. In another embodiment, lentiviral vectors are can beused as agents for the direct delivery and sustained expression of atransgene in several tissue types, including brain, retina, muscle,liver and blood. The vectors can efficiently transduce dividing andnondividing cells in these tissues, and maintain long-term expression ofthe gene of interest. For a review, see, Naldini, Curr. Opin.Biotechnol., 9:457-63, 1998; see also Zufferey, et al., J. Virol.,72:9873-80, 1998). Lentiviral packaging cell lines are available andknown generally in the art. They facilitate the production of high-titerlentivirus vectors for gene therapy. An example is atetracycline-inducible VSV-G pseudotyped lentivirus packaging cell linewhich can generate virus particles at titers greater than 106 IU/ml forat least 3 to 4 days (Kafri, et al., J. Virol., 73: 576-584, 1999). Thevector produced by the inducible cell line can be concentrated as neededfor efficiently transducing nondividing cells in vitro and in vivo.

Non-viral vectors. A vector can be introduced in vivo in a non-viralvector, e.g., by lipofection, with other transfection facilitatingagents (peptides, polymers, etc.), or as naked DNA. Synthetic cationiclipids can be used to prepare liposomes for in vivo transfection, withtargeting in some instances (Felgner, et. al., Proc. Natl. Acad. Sci.U.S.A. 84:7413-7417, 1987; Felgner and Ringold, Science 337:387-388,1989; see Mackey, et al., Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031,1988; Ulmer et al, Science 259:1745-1748, 1993). Useful lipid compoundsand compositions for transfer of nucleic acids are described inInternational Patent Publications WO95/18863 and WO96/17823, and in U.S.Pat. No. 5,459,127. Other molecules are also useful for facilitatingtransfection of a nucleic acid in vivo, such as a cationic oligopeptide(e.g., International Patent Publication WO95/21931), peptides derivedfrom DNA binding proteins (e.g., International Patent PublicationWO96/25508), or a cationic polymer (e.g., International PatentPublication WO95/21931). Recently, a relatively low voltage, highefficiency in vivo DNA transfer technique, termed electrotransfer, hasbeen described (Mir et al., C.P. Acad. Sci., 321:893, 1998; WO 99/01157;WO 99/01158; WO 99/01175). DNA vectors for gene therapy can beintroduced into the desired host cells by methods known in the art,e.g., electroporation, microinjection, cell fusion, DEAE dextran,calcium phosphate precipitation, use of a gene gun (ballistictransfection), or use of a DNA vector transporter (see, e.g., Wu et al.,J. Biol. Chem. 267:963-967, 1992; Wu and Wu, J. Biol. Chem.263:14621-14624, 1988; Hartmut et al., Canadian Patent Application No.2,012,311, filed Mar. 15, 1990; Williams et al., Proc. Natl. Acad. Sci.USA 88:2726-2730, 1991). Receptor-mediated DNA delivery approaches canalso be used (Curiel et al., Hum. Gene Ther. 3:147-154, 1992; Wu and Wu,J. Biol. Chem. 262:4429-4432, 1987). U.S. Pat. Nos. 5,580,859 and5,589,466 disclose delivery of exogenous DNA sequences, free oftransfection facilitating agents, in a mammal.

EXAMPLES

The present invention will be better understood by reference to thefollowing examples, which are provided by way of exemplification and arenot intended to limit the invention.

Example 1 Identification and Characterization of NRIF3

To further our understanding of the molecular events underlyingreceptor-activated transcription, we sought to identify additionalco-activators using a yeast two-hybrid screening strategy (29). ThisExample describes the isolation of a novel co-activator for nuclearreceptors, designated as NRIF3. Fluorescence microscopy indicates thatNRIF3 is a nuclear protein. The yeast two-hybrid and in vitro bindingassays revealed that NRIF3 interacts specifically with TR and RXR in aligand-dependent fashion but does not interact with other examinednuclear receptors. Transfection studies indicate that NRIF3 selectivelypotentiates TR- and RXR-mediated transactivation in vivo. NRIF3 encodesa small protein of 177 amino acids and other than an N-terminal LxxLL(SEQ ID NO:1) motif shares no homology with known co-activators. Thecombination of computer modeling, domain and mutagenesis analysessuggest that NRIF3 interacts with nuclear receptors through itsC-terminal domain that contains a novel LxxIL (SEQ ID NO:2) module,while other part of NRIF3 may contribute to its observed receptorspecificity. These findings may provide novel insight into the molecularmechanism(s) of receptor-mediated transcriptional activation as well asthe functional specificity of nuclear receptors.

Materials and Methods

Isolation of NRIFs and the yeast two-hybrid assay. The Brent two-hybridsystem (29) was employed to isolate candidate cDNA clones interactingwith LexA-TRα in a ligand-dependent fashion. Full length chicken TRα(cTRα) was fused in frame to the C-terminus of the LexA DBD in pEG202(29). The LexA-TRα bait, the LacZ reporter (pSH18-34), and a pJG4-5based HeLa cell cDNA library were transformed into the yeast strainEGY48 (29). The transformants were selected on Gal/Raf/X-gal medium inthe absence of leucine and were further screened for the expression ofLacZ in the presence of 1 μM T3. Blue colonies were picked andre-examined for T3-dependent expression of LacZ. Positive yeast cloneswere then selected and plasmids harboring candidate prey cDNAs wereisolated. Individual candidate prey plasmid was then amplified in E.coli and re-transformed into the original yeast strain to confirm theinteraction phenotype. The cDNA inserts were then sequenced using anautomatic sequencer. Four novel clones (NRIF1, 2, 3, and 4) wereobtained. Among them, NRIF3 was a full length clone.

Wild type NRIF3, the endonexin long form (EnL) and short form (EnS), andthe L9A NRIF3 mutant were examined for their interaction with variousnuclear receptors in a yeast two-hybrid assay. The following receptorbaits were used: LexA-cTRα LBD, LexA-hTRβ LBD, LexA-hRARα LBD,LexA-hRXRα LBD, and LexA-hGR LBD. The NRIF3 C-terminal domain (NCD) wasfused in frame with the LexA DBD and examined for interaction withreceptor LBDs using the following preys: B42-cTRα LBD, B42-hRARα LBD,and B42-hRXRα LBD expressed from pJG4-5. Yeast cells harboringappropriate plasmids were grown in selective media with Gal/Raf in thepresence or absence of cognate ligand (1 μM T3 for TR, all trans or9-cis RA for RAR, 9-cis RA for RXR, and 10 μM deoxycorticosterone forGR) overnight before β-galactosidase activity was assayed usingo-Nitrophenyl β-D-Galactopyranoside as the substrate. β-galactosidaseunits are expressed as (O.D. 420 nm×1000)/(minutes of incubation×O.D.600 nm of yeast suspension).

Fluorescence Microscopy. Full length NRIF3 was cloned into the GFPfusion protein expression vector pEGFP (Clontech). The resultingGFP-NRIF3 vector and the control plasmid pEGFP were transfected intoHeLa cells by calcium phosphate co-precipitation. Cells were incubatedat 37° C. for 24 hours before the examination with a fluorescencemicroscope to determine the subcellular location of GFP-NRIF3 or the GFPcontrol.

In vitro binding assay. Full length NRIF3 was cloned into pGEX2T, abacterial GST-fusion protein expression vector (Pharmacia). TheGST-NRIF3 fusion protein was expressed in E. coli and affinity purifiedusing glutathione-agarose beads (30). ³⁵S-labeled full length cTRα,hRARα, hRXRα, hVDR, hGR, hPR, and hER were generated by in vitrotranscription/translation using a reticulocyte lysate system (Promega).Binding was performed as previously described (30), using the followingbuffer: 20 mM Hepes (pH 7.9), 1 mM MgCl₂, 1 mM DTT, 10% Glycerol, 0.05%Triton X-100, 1 μM ZnCl₂, and 150 mM KCl. Appropriate ligands were addedinto the binding reaction when indicated: 1 μM T3 for TR, 1 μM all transRA or 9-cis RA for RAR, 1 μM 9-cis RA for RXR, and 150 nM 1,25-(OH)₂VitD3, dexamethathone, progesterone, or estradiol for VDR, GR, PR, orER. After the binding reaction, the beads were washed three times andthe labeled receptors bound to the beads were examined in 10% SDS-PAGEfollowed by autoradiography. Five percent of the ³⁵S-labeled receptorinput was also electrophoresed in the same gel.

Transfection studies. Most reporters used in this study have beendescribed previously, including IR-ΔMTV-CAT, DR4-ΔMTV-CAT,GH-TRE-tk-CAT, IR+3 (ERE)-ΔMTV-CAT (5, 25, 78). A DR1-ΔMTV-CAT reporterresponsive to RXR was obtained from Ron Evans. A GRE/PRE-tk-CAT reporterwas obtained from Gunther Schutz. The (IR)2-TATA-CAT was constructed inour laboratory by cloning two copies of the IR sequence (AGGTCA TGACCT)upstream of a TATA element derived from the tk promoter. A hVDRexpression vector and the VDRE-ΔMTV-CAT containing the VDRE from theosteocalcin promoter were obtained from J. Wesley Pike. Vectorsexpressing cTRα, hRARα, hRXRα, rGR, hPR, and hER have been describedpreviously (17, 25, 26, 50, 53, 81). The NRIF3 expression vector wasconstructed by cloning full length NRIF3 into a pExpress vector (25).Appropriate plasmids were transfected into HeLa cells by calciumphosphate co-precipitation using 25-100 ng of the receptors, 250-500 ngof the CAT reporters, and 750 ng of the NRIF3 or control pExpressvector. After transfection, cells were incubated at 37° C. (with orwithout cognate ligands) for 42 hours before being harvested. CAT assayswere carried out as previously described (30). Relative CAT activity wasdetermined as the percent acetylation of substrate per 30 μg of cellprotein in a 15 hour incubation at 37° C. The results were calculatedfrom duplicate or quadruplicate samples and the variation among sampleswas less than 10%.

Domain and mutagenesis analyses. To construct pJG4-5 derived vectorsexpressing EnL or EnS, the pJG4-5/NRIF3 plasmid was digested with NcoIand XhoI, and the resulting vector fragment was gel-purified. Thisfragment was then ligated to an EnL or EnS insert generated frompExpress-EnL or pExpress-EnS by an NcoI/SalI double digest. Theresulting pJG4-5/EnL or EnS plasmids were confirmed by sequenceanalysis. The L9A mutant form of NRIF3 was generated by site-directedmutagenesis using a PCR-based method, and the mutation was confirmed bysequence analysis. pJG4-5 derived vectors expressing EnL, EnS, or theL9A NRIF3 mutant form were transformed into yeast strains harboring theLacZ reporter (pSH18-34) and appropriate bait plasmids (LexA-TR,LexA-RAR, LexA-RXR, and LexA-GR). Transformants were subjected toquantitative assays of β-galactosidase activity as described earlier.

To construct the bait plasmid expressing LexA-NCD, a derivative ofpEG202 (which contains a new polylinker) plasmid was digested with NcoIand XhoI and ligated to synthetic oligonucleotides that encode the last16 amino acids of NRIF3 (residues 162-177). Similarly, mutant NCD wasgenerated by using oligonucleotides that contain the designed mutationsin the ligation reaction. All constructs were confirmed by sequenceanalysis. Bait plasmids expressing LexA-NCD or LexA-mutant NCD weretransformed together with one of these prey plasmids (B42-TR LBD,B42-RXR LBD, and B42-RAR LBD) into the yeast strain that harbors theLacZ reporter (pSH18-34). Subsequent two-hybrid assays were carried outas described earlier.

Docking of co-activator peptides to receptors. We built a model of theinteraction between the 17-residue C-terminal peptide of NRIF3(KASRHLDSYEFLKAILN; SEQ ID NO:7) and the LBDs of several receptors (TRαwas used as an example in FIG. 10). An LxxIL (SEQ ID NO:2) motif withinthe NRIF3 peptide is underlined. A similar modeling procedure wascarried out on a 20-residue peptide (SLTERHKILHRLLQEGSPSD; SEQ ID NO:8)of the second LxxLL (SEQ ID NO:1) box of SRC-1 (52). We hypothesizedthat the LxxIL motif of the C-terminus of NRIF3 contacts theco-activator binding site of the nuclear receptors, and the automaticdocking procedure was carried out towards this site (71, 75, 76). Twocritical features of the interaction between the LBDs of nuclear hormonereceptors and their co-activators were used to build the models: 1) The“charge clamp”, initially observed in the complex between SRC-1 andPPARγ (56), where a conserved glutamate and lysine at opposite ends ofthe hydrophobic cavity of the receptors contact the backbone of theco-activator's LxxLL box. This feature enabled the orientation of theNRIF3 helical peptide and, 2) The finding that the leucines of the LxxLLmotif of SRC-1 are buried into the hydrophobic cavity of the receptor.This feature makes predictions of the side of the NRIF3 peptide whichfaces the receptor.

The co-activator peptides were assigned a helical secondary structure,the backbone φ and ψ angles being −62 and −41 degrees, respectively. Theω angle was set to 180 degrees. Loose distance restraints were setbetween the “charge clamp” of the receptors (56) and C^(α) atoms of thepeptide. The energy of the complex was minimized in the internalcoordinate space using the modified ECEPP/3 potentials. The subset ofthe variables minimized with the ICM method (1, 71, 76), included theside-chains of the receptor, six positional variables of the helix andthe side-chain torsion angles of the helix.

Binding energy calculation. The binding energy was calculated by thepartitioning method as described elsewhere (64). Briefly, the bindingenergy function is partitioned into three terms: the surface (orhydrophobic) term, determined as the product of the solvent accessiblesurface by a surface tension of 30 cal/mol/Å², the electrostatic term,calculated by a boundary element algorithm, with a dielectric constantof 8, and the entropic term, which results from the decrease inconformational freedom of residue side-chains partially or completelyburied upon complexation.

Results

Cloning of NRIF3 cDNA. To isolate potential co-activators mediating thetranscriptional activation function of nuclear receptors, we employed ayeast two-hybrid screening strategy (29). A bait expressing a fulllength TRα fused to the C-terminus of the LexA DNA binding domain wasused to screen a HeLa cell cDNA library cloned into pJG4-5 (29).Candidate clones that exhibited a thyroid hormone (T3)-dependentinteraction with LexA-TRα were selected and further examined andsequenced. Four novel clones were identified and all were found toexhibit similar interaction with the ligand binding domain (LBD) of TRαas with full length receptor (data not shown). These clones weredesignated as NRIF1, 2, 3 and 4 (Nuclear receptor interacting factors).Not surprisingly, the LBD of TRb was also found to interact with theseNRIFs in a T3-dependent manner (data not shown). Among these fourisolated NRIFs, NRIF3 was a full length clone. As shown in FIG. 1, LexAalone (negative control) does not interact with NRIF3 (as indicated bythe low β-galactosidase activity) and incubation with T3 has no effect.Similarly, no interaction was detected between the LexA-TR LBD and B42alone with or without T3 (data not shown). The LexA-TR LBD also showslittle interaction with NRIF3 in the absence of T3. However, incubationwith T3 results in strong stimulation of the NRIF3-TR LBD interaction(FIG. 1). The extent of T3-dependent interaction between NRIF3 andLexA-TR LBD was similar to that of Trip1 (FIG. 1), one of the firstthyroid hormone receptor interacting factors cloned using a two-hybridscreen (42).

Sequence analysis of NRIF3. Sequence analysis of the NRIF3 cDNA revealeda single open reading frame (ORF) encoding a polypeptide of 177 aminoacids (FIG. 2). NRIF3 shares no homology with members of the SRC-1 andCBP/p300 families. The size of NRIF3 is in sharp contrast to the size ofCBP/p300 (around 300 kd), or the SRC-1 family (around 160 kd). NRIF3contains a putative nuclear localization signal (KRKK; (SEQ ID NO:5), aswell as one copy of an LxxLL (SEQ ID NO:1) motif (amino acids 9-13) thatwas recently identified to be essential for the interaction of a numberof putative co-activators with nuclear receptors (32).

A database search identified two highly-related homologs of NRIF3, whichwere previously designated as β3-endonexin short form and long form(67). The endonexin short form (EnS) was originally isolated from atwo-hybrid screen intended to clone factors that interact with thecytoplasmic tail of integrin β3 (67). The long form (EnL) was thenidentified as an alternatively spliced product of the same gene.However, the long form does not bind to integrin β3 (67). Nucleotidesequence comparisons between cDNAs of NRIF3 and endonexin short or longforms indicate that NRIF3 is a third alternatively spliced product ofthe same gene (alignment not shown). The precise function(s) of the twoendonexin proteins is currently under investigation.

NRIF3 localizes to the cell nucleus. Although a putative nuclearlocalization signal was found in NRIF3, we considered it important toidentify the subcellular location of the NRIF3 protein since extensivehomology was found between NRIF3 and the two endonexins. The entireNRIF3 ORF was fused to the C-terminus of green fluorescent protein (GFP)(18). The resulting GFP-NRIF3 fusion protein was expressed in HeLa cellsby transient transfection and the subcellular location of the fusionprotein was visualized by fluorescence-microscopy. As shown in FIG. 3,the control GFP protein is distributed throughout the cell whileGFP-NRIF3 is localized exclusively to the nucleus. This result suggeststhat NRIF3 is a nuclear protein, which is compatible with its putativerole as a nuclear receptor co-activator.

Selective interaction of NRIF3 with liganded nuclear receptors in yeast.Although NRIF3 was originally cloned using full length TRα as the bait,we later identified that the region of the receptor responsible forNRIF3 binding is its LBD (see FIG. 1). A common feature among most ofthe known co-activators that show ligand-dependent interaction withnuclear receptors is the presence of the LxxLL (SEQ ID NO:1) motif(s) intheir receptor interaction domains. The LxxLL motif appears to beinvolved in direct contact with a structurally-conserved surface in theligand-bound LBDs of the receptors (23), which may provide the molecularbasis for the broad spectrum of receptor binding by co-activators suchas SRC-1 or GRIP1. Since a putative LxxLL motif is also present in NRIF3(amino acids 9-13), we asked whether NRIF3 also interacts with the LBDsof other nuclear receptors.

The LBDs of several nuclear receptors were examined for interaction withNRIF3 in a yeast two hybrid assay. As shown in Table 1, NRIF3 does notinteract with LexA alone (negative control) with (+) or without (−)ligand. LexA-TR and LexA-RXR show little (if any) interaction with NRIF3in the absence of their cognate ligands. However, the presence of T3(for TR) or 9-cis RA (for RXR) results in a strong stimulation of theirinteraction with NRIF3, as indicated by the induction of β-galactosidaseactivity (Table 1). Interestingly, when LexA-RAR or LexA-GR was used asthe bait, no interaction was detected with NRIF3 in the presence orabsence of their cognate ligands (Table 1). The finding that NRIF3interacts with TR but not RAR was surprising in light of a recent study,which shows that TR and RAR functionally interact with the same LxxLL(SEQ ID NO:1) boxes (boxes 2 and 3) of SRC-1/NCoA-1 (52). As positivecontrols, we confirmed that both LexA-RAR and LexA-GR exhibitedligand-dependent interaction with other co-activators that are notreceptor-specific (data not shown). Taken together, these resultssuggest that NRIF3 exhibits differential specificity in its interactionwith different nuclear receptors.

TABLE 1 Interaction of NRIF 3 with Nuclear Receptors in Yeastβ-galactosidase activity Ligand Bait Prey − + Fold Stimulation LexANRIF3-B42 2.3 1.9 0.8 LexA-TR NRIF3-B42 1.8 125 69 LexA-RAR NRIF3-B420.1 0.1 1 LexA-RXR NRIF3-B42 0.2 63 315 LexA-GR NRIF3-B42 0.8 0.6 0.8The LacZ reporter activity was determined for yeast strains harboringthe indicated bait and prey plasmids in the presence of (+) or absence(−) of cognate ligands as described in Materials and Methods. See textfor detailed explanations.

NRIF3 specifically binds to TR and RXR but not to other nuclearreceptors in vitro. To further examine the interaction between NRIF3 andvarious nuclear receptors as well as to confirm the potential receptorspecificity of NRIF3, in vitro GST binding assays were performed (30).³⁵S-labeled nuclear receptor, generated by in vitrotranscription/translation, was incubated with purified GST-NRIF3 or theGST control bound to glutathione-agarose beads. All binding assays werecarried out with (+) or without (−) the cognate ligand of the examinedreceptor. As shown in FIG. 4 (top left), TR and NRIF3 interact poorly inthe absence of T3. Addition of T3 results in a strong increase in TRbinding to GST-NRIF3, confirming that NRIF3 associates with TR in aT3-dependent manner. Using similar binding assays, we also studied theinteraction of NRIF3 with six other nuclear receptors. Consistent withour findings from the yeast two-hybrid studies (Table 1), NRIF3interacts with RXR in vitro in a ligand-dependent manner (FIG. 4), butshows little or no binding to other nuclear receptors (RAR, VDR, GR, PR,and ER) in the presence or absence of their cognate ligands (FIG. 4).Taken together, the results of the yeast two-hybrid (Table 1) and the invitro binding (FIG. 4) assays suggest that NRIF3 possesses a distinctreceptor specificity.

NRIF3 Selectively Potentiates TR- and RXR-Mediated Transactivation inVivo. To examine the potential role of NRIF3 in TR-mediatedtransactivation, transfection studies were carried out. HeLa cells,which lack endogenous TR (25), were transfected with a vector expressingTR, and a CAT reporter under the control of the ΔMTV basal promoterlinked to an idealized inverted repeat (IR) (AGGTCATGACCT; (SEQ ID NO:9)TRE sequence (IR-ΔMTV-CAT) (25), along with either a control plasmid ora vector expressing NRIF3. As shown in FIG. 5A, NRIF3 significantlyenhances TR-mediated activation of the CAT reporter (typically 2.5- to3-fold). As a control, we also examined the effect of CBP, a reportedco-activator for nuclear receptors (13, 37), and found that itsexpression results in a similar degree of enhancement as with NRIF3(around 3-fold) (FIG. 5A).

We also examined another CAT reporter controlled by the Herpes virusthymidine kinase (tk) promoter linked to native rat growth hormone (GH)TRE sequences (5). NRIF3 was found to also enhance TR-mediatedactivation of this reporter (about 3.5-fold) (FIG. 5B). In addition,using similar transfection assays, we found that NRIF3 enhancesTR-mediated activation of two other reporters, (IR)2-TATA-CAT andDR4-ΔMTV-CAT (data not shown). Therefore, NRIF3 potentiates TR-mediatedtransactivation in a variety of different TRE/promoter contexts. Takentogether, the results of these transfection studies suggest that NRIF3can function as a co-activator of TR.

To examine whether NRIF3 can also act as a co-activator for RXR, HeLacells were transfected with the IR-ΔMTV-CAT reporter, whose IR sequencecan also function as a strong response element for the RXR(s) and RAR(s)(25, 49, 61). HeLa cells express endogenous RXR(s) and RAR(s), as theactivity of the IR-ΔMTV-CAT reporter is strongly stimulated by theircognate ligands, even without co-transfection of any receptor expressionplasmid (FIG. 6A, panels 1, 3, and 5). Co-transfection of NRIF3 enhancesthe activation of this reporter by either 9-cis RA, or LG100153 (72), anRXR-specific ligand (FIG. 6A, panels 1 and 2; 3 and 4). In contrast,although the RAR-specific ligand TTNPB (68) also activates theIR-ΔMTV-CAT reporter, co-transfection of NRIF3 has no effect (FIG. 6A,panels 5 and 6). These results indicate that NRIF3 potentiates theactivity of endogenous RXR(s) but not RAR(s), which is consistent withthe distinct receptor specificity of NRIF3 revealed from the yeasttwo-hybrid assay (Table 1) and in vitro binding studies (FIG. 4).

To further document that NRIF3 can function as a co-activator for RXR, avector expressing exogenous RXR was co-transfected with IR-ΔMTV-CAT.Exogenous RXR expression enhances the activation of this CAT reporter byeither 9-cis RA or LG100153 (comparing FIGS. 6B and 6A, panels 1 and 3).This RXR-mediated activation of reporter expression is furtherstimulated by NRIF3 (FIG. 6B). Finally, we also examined the activationof a DR1-ΔMTV-CAT reporter. This DR1 (AGGTCAnAGGTCA; SEQ ID NO:10)sequence is thought to be a specific response element for RXR (39, 51).In the sequence, n represents any nucleotide. Although we found thatthis DR1 is a weaker response element than the IR sequence,co-transfection of an RXR expression vector leads to ligand-inducedactivation of this DR1 reporter, which is also further enhanced by NRIF3(FIG. 6C).

NRIF3 does not potentiate the activity of GR, PR, ER, and VDR in vivo.The selective co-activation of TR and RXR (but not RAR) by NRIF3 isconsistent with its distinct binding specificity to these receptors. Tofurther establish that NRIF3 acts as a receptor-specific co-activator,we next examined the effect of NRIF3 on the activity of four additionalnuclear receptors, including GR, PR, ER, and VDR, by transfectionstudies. HeLa cells were transfected with a GRE/PRE-tk-CAT reporteralong with a vector expressing either GR or PR. As shown in FIG. 7A,cognate hormone treatment results in activation of the CAT reporter.However, expression of NRIF3 has little effect (FIG. 7A). Similarexperiments were carried out using ER and ERE-ΔMTV-CAT, or VDR andVDRE-ΔMTV-CAT. As shown in FIGS. 7B and 7C, NRIF3 was found to havelittle or no effect on the activity of these receptors as well. Takentogether, the combined results of our transfection studies support thenotion that NRIF3 is a co-activator with a unique receptor specificity.

A novel C-terminal domain in NRIF3 is essential for ligand-dependentinteractions with TR and RXR. The LxxLL (SEQ ID NO:1) signature motifhas been found to be present in the receptor interacting domain of manyidentified co-activators such as SRC-1/NCoA-1 and GRIP1/TIF-2 (32). Thebroad spectrum of receptor binding by co-activators such as SRC-1suggests that the LxxLL-containing interacting domain may recognize astructurally-similar surface of these LBDs. Indeed, recent structuraland functional studies revealed that the LxxLL motif and its nearbyflanking amino acids are involved in direct contact with a hydrophobiccleft of the target surface presented by the ligand-bound LBDs ofnuclear receptors (19, 23, 52, 56). The fact that NRIF3 also contains anLxxLL motif (amino acids 9-13, see FIG. 2 and FIG. 8A) and exhibits adistinct receptor specificity, raises the possibility that: 1), themotif and surrounding amino acids are involved in mediatingreceptor-specific interaction of NRIF3; or, 2) another region of NRIF3(alone or in concert with the LxxLL motif region) plays an importantrole in mediating such interaction.

To explore these questions, we examined whether the endonexin short form(EnS) and endonexin long form (EnL), which contain the same LxxLL (SEQID NO:1) motif and flanking amino acids as NRIF3, can interact withnuclear receptors in a yeast two-hybrid assay (FIG. 8). EnS consists of111 amino acids and is 100% identical to the first 111 residues ofNRIF3, while the first 161 amino acids of EnL (170 amino acids) is also100% identical to the same region in NRIF3 (see FIG. 2 legend and FIG.8A). Thus, NRIF3 and EnL differ only in their C-terminus, with a uniqueregion of 16 amino acids in NRIF3 or 9 residues in EnL (FIG. 8A).Interestingly, despite their extensive identity with NRIF3, theinteraction with liganded TR or RXR is completely abolished in EnS andEnL (FIG. 8B). We also examined other nuclear receptors that do notinteract with NRIF3 and found that they also do not interact with EnS orEnL (data not shown). These results indicate that the unique C-terminaldomain in NRIF3 (residues 162-177) is essential for its specificinteraction with liganded TR and RXR, while the N-terminal LxxLL motif(amino acids 9-13) and its flanking sequences are not sufficient toallow for detectable receptor interactions.

Although found to be not sufficient for interaction, we examined whetherthe N-terminal LxxLL motif of NRIF3 contributes in the NRIF3/receptorinteraction by mutating the first leucine of the LxxLL motif intoalanine (L9A) by site-directed mutagenesis. Previous studies have shownthat the three leucine residues are essential for an LxxLL module tointeract with receptor LBDs, and the replacement of any of them withalanine would abolish the interaction (32). We examined the mutantNRIF3(L9A) for its interaction with TR and RXR in a yeast two-hybridassay. As shown in FIG. 9, the L9A mutant is still capable ofligand-dependent interaction with TR and RXR (¹⁸25-fold induction byligand). However, the introduced mutation reduces the interaction byabout 4-fold (for TR) or 14-fold (for RXR). These results suggest thatalthough the LxxLL motif is not absolutely essential for NRIF3interaction with liganded receptors, it plays a role in allowing anoptimum interaction to occur.

Computer modeling suggests that the C-terminal domain of NRIF3 docksinto the hydrophobic cleft of the liganded LBDs. Secondary structureanalysis of the C-terminal domain of NRIF3 predicts the formation of anα-helix. Moreover, inspection of the putative C-terminal helix revealedan LxxIL (SEQ ID NO:2) motif (amino acids 172-176), which is reminiscentof the canonical LxxLL (SEQ ID NO:1). Although the ultimate elucidationof the molecular basis of the NRIF3-receptor interaction awaits futurestudies such as X-ray crystallography, the putative helix structure ofthe NRIF3 C-terminal domain and its LxxIL motif suggest that it mayinteract with the liganded LBDs in a similar fashion as to thereceptor-interacting domains that employ the canonical LxxLL motif. Toexplore this possibility, we modeled the interaction of the C-terminusof NRIF3 with the liganded LBDs, using algorithms developed mainly bythe laboratory of one of the authors (R. Abagyan and co-workers) (1, 63,70, 74, 75). The background information and procedures used forconstructing these models are described in Materials and Methods. Theresults of our modeling suggest that the NRIF3 C-terminal domain(referred as NCD) fits well into the hydrophobic cleft formed on theLBDs as a result of ligand binding. An example of such a model (NCD/TRLBD) is shown in FIG. 10. In this model, the two leucines and oneisoleucine (green and cyan) of the LxxIL motif are predicted to bedeeply buried into the central cavity of the hydrophobic groove formedby the liganded LBD of the receptor. We also calculated the putativebinding energy for the modeled NCD/TR complex, using an improvedpartitioning binding energy function, with continuum representation ofthe electrostatics of the system (64). The calculated binding energy forthe modeled NCD/TR complex is about −21 kcal/mol. As a control, wecarried out a similar modeling procedure using the second LxxLL boxwithin the receptor interacting domain of SRC-1. This LxxLL box has beenshown to be required for interaction with TR (52). Our calculatedbinding energy for this LxxLL box with liganded TR LBD is −18 kcal/mol,a value that is very close to the one calculated for the NRIF3C-terminal domain. Altogether, our modeling and calculations suggest amechanism in which the C-terminal domain of NRIF3 directly mediatesinteraction with liganded LBDs through an LxxIL motif.

Functional interaction of the NRIF3 C-terminal domain with liganded LBDsand the essential role of its LxxIL motif. To explore the possibilitysuggested from our computer modeling, the C-terminal domain of NRIF3(amino acids 162-177, referred as NCD) was fused to the LexA DNA bindingdomain and was examined for interaction with the receptor LBDs in ayeast two-hybrid assay. The LexA-NCD fusion protein alone does notactivate the LacZ reporter in yeast (data not shown). As a negativecontrol, we also found that LexA-NCD does not interact with the B-42activation domain itself (FIG. 11), and LexA alone does not interactwith the receptor LBDs (data not shown). However, when the LexA-NCD andthe LBD of TR or RXR (fused with B-42) were used in the two-hybridassay, a strong ligand-dependent interaction was observed, as indicatedby the induction of β-galactosidase activity by their cognate ligands(FIG. 11). These results suggest that the NRIF3 C-terminal domain candirectly interact with the LBDs of TR and RXR in a ligand-dependentmanner.

Since NRIF3 harbors a distinct receptor specificity in interacting onlywith TR and RXR but not other receptors (e.g. RAR), we next askedwhether the NCD also harbors a receptor specificity. To our surprise,the NCD was found to interact efficiently with the LBD of RAR in aligand-dependent manner (FIG. 11). Therefore, while our results clearlysuggest that the NCD is an important surface for receptor interactions,as the NCD is found to be both essential for (FIG. 8) and sufficient tomediate such interactions (FIG. 11), it nevertheless does not appear tobe (solely) responsible for the receptor specificity of NRIF3. It ispossible that another region of the NRIF3 molecule may contribute to theobserved receptor specificity of NRIF3, and/or, the specificity isdetermined by the overall three-dimensional structure of NRIF3.

Since our model predicts the importance of the LxxIL (SEQ ID NO:2) motifin the NCD-receptor interaction (FIG. 10), we tested this by changingthe three core residues of the motif (two leucines and one isoleucine)into alanine. As expected, interaction with the LBDs is completelyabolished in the resulting mutant NCD (FIG. 11), confirming that theLxxIL motif is essential for the interaction.

Discussion

Recent efforts in understanding receptor-mediated transcription have ledto the identification of a number of co-activators for nuclear hormonereceptors, which can be categorized into two main groups based onoverall homology, the SRC-1 family (including SRC-1/NCoA-1,TIF2/GRIP1/NCoA-2, AIB1/p/CIP/ACTR/RAC3/TRAM-1) (2, 14, 34, 35, 37, 44,58, 73, 74, 79), and the CBP/p300 family (13, 31, 37). Other putativeco-activators (e.g. ARA70 and PGC-1) that do not belong to the SRC-1 orCBP/p300 families have also been identified (60, 85). In addition, p/CAFmay also be involved in receptor action through its association withnuclear receptors as well as with other co-activators (11, 14, 38, 83).Among these known co-activators, CBP/p300, members of the SRC-1 group,and p/CAF all possess HAT activities (8, 14, 57, 69, 83).

In this study we report the identification of a novel nuclear protein(NRIF3) which exhibits specific ligand-dependent interactions with TRand RXR but not with RAR, VDR, GR, PR, and ER. Functional studiesindicate that NRIF3 potentiates TR- and RXR-mediated transactivation invivo while it exhibits little or no effect on the activity of otherexamined receptors. Therefore, NRIF3 represents a novel co-activatorwith a distinct receptor specificity and, thus, may shed light onclarifying the molecular mechanism(s) underlying receptor-specificregulation of gene expression.

A database search indicated that NRIF3 shares no homology with any knownco-activators except for a single LxxLL (SEQ ID NO:1) motif. An unusualfeature of NRIF3 is its relatively small size, which is in sharpcontrast to SRC-1 or CBP/p300. A homology search identified twoalternatively spliced isoforms of NRIF3 which were previously designatedas β3-endonexin short and long forms (67). Preliminary studies withthese two endonexins indicate that, like NRIF3, they also localize tothe cell nucleus (S. Shattil, personnel communication; Li and Samuels,unpublished data). Interestingly, despite their extensive identitieswith NRIF3, both the endonexin short and long forms fail to exhibitinteraction with liganded nuclear receptors (see FIG. 8). Consistentwith this finding, we found that the endonexin short and long forms havelittle effect on receptor-mediated transcription in transfection studies(data not shown). Therefore, the precise roles of these two endonexinsremain to be elucidated. We suggest two not mutually-excludingpossibilities. First, since both the endonexin long and short formsappear to localize to the nucleus, it is possible that they may act asco-factors for other transcriptional regulators. Second, since theendonexin short form can interact with the cytoplasmic tail ofβ3-integrin (22, 67), it may function to communicate signals generatedat the plasma membrane to the cell nucleus. An example of a proteinwhich is involved in both cell adhesion and transcriptional regulationis β-catenin (82).

Previous study of the endonexins identified the presence ofNRIF3-related mRNAs (by Northern blots) in a wide range of human tissues(67). Because NRIF3 and the endonexin long form contain almost identicalnucleotide sequences and differ only by an alternative splice whichresults in the removal of a small exon in NRIF3, it is difficult tospecifically identify NRIF3 mRNA by Northern blots. A search of theexpressed sequence tag database indicates that NRIF3 as well as both theendonexin long and short form mRNAs are expressed. However, the precisedetermination of cell and tissue distribution of the individual NRIF3and endonexin short and long forms will require the development ofhighly selective antibodies. Nevertheless, the wide expression patternof NRIF3-related mRNAs is consistent with the role of NRIF3 as aco-activator of the TRs, which are also widely expressed (70), or theRXRs, which are ubiquitously expressed (48).

A key question concerning the action of nuclear hormone receptors is toelucidate the molecular events underlying the functional specificity ofdifferent receptors in regulating the expression of their target genes.Determinants of specificity include specific ligand binding, selectivebinding of the receptors to their cognate response elements, as well asspecific expression pattern of different receptors. These determinantsalone, however, are not always sufficient to explain the extent ofspecificity observed for members of the nuclear receptor family. Forexample, several members of the thyroid hormone/retinoid receptorsubfamily may bind similarly to common DNA elements while target genescontaining those elements are only selectively activated by certainreceptors (20, 47). Therefore, it is likely that additional factors(determined by cell/promoter contexts) are involved in determiningreceptor functional specificity. In this respect, most knownco-activators do not appear to be receptor-specific. For example,members of the SRC-1 family and CBP/p300 interact with and appear to beinvolved in the action of many nuclear receptors (13, 14, 34, 37). Twoknown co-activators that may be involved in receptor-specific functionsare ARA70 and PGC-1. The androgen receptor co-activator ARA70 has beenreported to potentiate the activity of AR more efficiently than forother nuclear receptors (85). However, whether ARA70 can associate withother receptors remains to be thoroughly examined. The expression ofPGC-1 is mainly restricted to the brown fat tissue and is thought to bedirectly involved in the regulation of thermogenesis by PPARγ (60).Nevertheless, PGC-1 exhibits a relatively broad spectrum of binding todifferent nuclear receptors. Therefore, the identification of NRIF3represents the first example of a co-activator with such aclearly-defined receptor specificity.

The receptor-specificity of NRIF3 raises an interesting question aboutits molecular mechanism. Domain analysis suggests that the LxxLL (SEQ IDNO:1) motif (amino acids 9-13) and its flanking sequences in NRIF3 arenot sufficient for interaction with liganded nuclear receptors. In fact,such interaction is completely abolished in the endonexin long form, analternatively spliced product which has the same LxxLL motif andcontains the first 161 amino acids (out of a total of 177 amino acids)of NRIF3. This result suggests that a putative domain consisting of thelast 16 amino acids of NRIF3 (residues 162-177) is essential for itsinteraction with liganded receptors. Inspection of this C-terminalregion of NRIF3 (NCD) indicates that it contains an LxxIL (SEQ ID NO:2)motif (amino acids 172-176) and secondary structure analysis predictsthe formation of an α-helix. The predicted helix structure and thesimilarity of LxxIL to the canonical LxxLL raise the possibility thatthis LxxIL-containing region may play a direct role in NRIF3-receptorinteractions.

Our modeling of the NCD-LBD interaction (FIG. 10) suggests that the samehydrophobic groove in the ligand-bound LBD, which has been shown byprevious studies to be the binding site for co-activators such asSRC-1/NCoA-1 or GRIP1 (19, 23, 56), appears also to be a suitable sitefor the docking of the C-terminal helix of NRIF3. Thus, the utilizationof the complementary pair of an α-helix (in the co-activator) and ahydrophobic groove (in the receptor) for interaction seems to be ageneral scheme that also applies to NRIF3. The binding energy estimatedfor the NCD and the TR LBD (−21 kcal/mol) is similar to the onecalculated for the second LxxLL box of SRC-1/NCoA-1 and the TR LBD (−18kcal/mol). To explore the mechanisms suggested by the modeling, we foundthat the NCD can directly mediate interaction with the LBDs in aligand-dependent manner (FIG. 11). Moreover, the LxxIL motif containedin the NCD was found to be essential for such interactions (FIG. 11). Insummary, the combination of computer modeling and domain/mutagenesisanalysis clearly suggest that the NCD is an important surface that isdirectly involved in interaction with the LBDs of the receptors, wherethe LxxIL motif of the NCD mimics the function of a canonical LxxLL. TheAF-2 helix (which is a critical constituent of the hydrophobic grooveformed upon ligand binding) of the LBD has been shown to be importantfor interaction with LxxLL boxes of the co-activators (23).Interestingly, we have examined two TR AF-2 mutants (66) and found thatin both cases, ligand-dependent interaction with NRIF3 was abolished (Liand Samuels, unpublished data).

However, the NCD alone does not appear to harbor the same specificity asNRIF3 (see FIG. 11). Thus, it seems likely that another part of theNRIF3 molecule may contribute to the observed specificity, and/or, thespecificity is determined by the overall three dimensional structure ofNRIF3. In this regard, the potential role of the N-terminal LxxLL (SEQID NO:1) motif is intriguing. Although the N-terminal LxxLL motif (aminoacids 9-13) is insufficient alone to mediate an interaction with TR orRXR (see FIG. 8), it can influence the interaction of NRIF3 with thesereceptors, as the NRIF3 L9A mutant retains significant but neverthelessreduced association with liganded TR or RXR (see FIG. 9). Thus, NRIF3appears to employ at least two regions in interacting with liganded TRor RXR, with the NCD playing an essential role and the N-terminal LxxLLmotif playing a secondary role. A simplified explanation for thesefindings would be that the NCD provides a major surface for receptorbinding, while the N-terminal LxxLL motif makes some minor contact witheither the same receptor molecule, or more likely, with the otherpartner of a homodimer or heterodimer to further stabilize theNRIF3-receptor interaction. An example of a co-activator moleculeemploying two separate regions to interact with the two partners of areceptor dimer has been documented in the recently solved crystalstructure of liganded PPARγ complexed with SRC-1/NCoA-1 (56). If NRIF3indeed employs both its NCD and its N-terminal LxxLL motif in receptorinteractions, the specificity may result from the intramolecular dialogbetween the two regions as well as the intermolecular dialog among NRIF3and the receptors. However, it remains possible that the N-terminalLxxLL may only play a more indirect role and the overallthree-dimensional structure of NRIF3 is responsible for its observedspecificity.

Accumulating evidence suggests that the actions of transcriptionalactivating proteins are (usually) mediated by multi-protein complexes(59) and such a scheme is also likely for nuclear receptors. Forexample, biochemical evidence suggests that multi-protein complexesassociate with liganded TR and VDR (24, 62, 86). Interestingly, many ofthe proteins identified in these studies are not known co-activators.While the study of known co-activators such as CBP/p300 and the SRC-1family has suggested that histone acetylation may play an important rolein receptor-mediated transactivation (8, 14, 57, 69), detailedelucidation of the transactivation mechanism(s) by these receptorsawaits the identification and study of additional co-factors involved inthe transactivation process.

Our results with NRIF3 suggest that transcriptional activation bynuclear receptors may employ receptor-specific co-activator(s) inaddition to the generally-utilized co-activators such as CBP or SRC-1.Therefore, co-activators with NRIF3-like properties may contribute tothe functional specificity of nuclear receptors in vivo. Based on ourresults with NRIF3 and previous studies of nuclear receptor action, wesuggest a “combinatorial specificity model” where a co-activationcomplex is likely composed of two kinds of factors: “general factors”that interact with and are involved in the action of many nuclearreceptors (such as CBP or SRC-1), and “specific factors” that exhibitreceptor specificity (such as NRIF3). In addition to their interactionwith the liganded receptor, co-activators may also communicate with eachother within the co-activation complex through protein-proteininteractions (e.g. CBP/p300 can interact with SRC-1/NCoA-1 or p/CIP)(37, 74, 84). An intriguing possibility is that the combinatorialactions of “specific factors” and other partners involved in thetransactivation process facilitate the recruitment of specificco-activation complexes for different receptors (under differentcell/promoter/HRE contexts), which would provide an importantmechanistic layer for receptor functional specificity. An advantage ofemploying such a combinatorial strategy is that a broad array ofdiversity can be generated from a relatively small number of involvedfactors. Further study of NRIF3 with known and possibly other yet to beidentified co-activators, as well as analysis of the interplay amongthese co-activators should provide important insights into the molecularmechanism(s) underlying the specificity of receptor-mediated regulationof target gene expression.

Example 2 Specific Antibodies Against NRIF3

Since alternative splicing of the NRIF3 gene generates multiple relatedmRNAs of similar sizes, it has been difficult to specifically identifythe tissue/cell expression pattern of NRIF3 by Northern blot. Tofacilitate the detection of NRIF3, we have developed specific antibodiesagainst NRIF3 protein. NRIF3 contains a unique C-terminal domain (aminoacids 162-177, referred as NCD) that is not present in otheralternatively-spliced products (known as endonexin short and longforms). Therefore, a polypeptide corresponding to the NCD wassynthesized, linked to a carrier and used to immunize rabbits (AlphaDiagnostic International, San Antonio, Tex.). To examine the specificityof the polyclonal antibodies obtained from the rabbits, known amounts ofpurified recombinant NRIF3 as well as endonexin short and long formswere used for a Western blot analysis. The result of this analysis showsthat less than 1 ng of the NRIF3 protein can be easily detected with ourantibodies, while no cross-reaction was observed between the NRIF3antibodies and the endonexin short or long forms even when more than athousand fold of these two proteins were used in the assay. Therefore,our NRIF3 antibodies appear to be both highly efficient and highlyspecific and should be an important tool in future studies involvingNRIF3.

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The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.

Patents, patent applications, and publications are cited throughout thisapplication, the disclosures of which are incorporated herein byreference in their entireties.

1. An isolated functional NRIF3 nuclear hormone receptor co-activatorpolypeptide (NRIF3 polypeptide), wherein the NRIF3 polypeptide binds ina ligand dependent manner to thyroid hormone receptor (TR) and retinoidX receptor (RXR), but does not interact with retinoic acid receptor(RAR), vitamin D receptor (VDR), progesterone receptor (PR),glucocorticoid receptor (GR), and estrogen receptor (ER) in a yeast twohybrid assay system or in vitro, or both, which polypeptide contains anLxxIL (SEQ ID NO:2) module in its C-terminal domain, and wherein theNRIF3 polypeptide exhibits at least about 85% identity to SEQ ID NO:4.2. The isolated functional NRIF3 polypeptide according to claim 1,comprising an amino acid sequence of SEQ ID NO:4 (FIG. 2).